A pretreatment method for blood analysis

Abstract: A pretreatment method for blood analysis comprising the step of selectively labeling leukocytes by treating blood sample with an aqueous solution comprising at least one surfactant selected from the group consisting of a cationic surfactant and an amphoteric surfactant, and with a labeling substance wherein; the aqueous solution of the surfactant is used at a concentration not to damage the whole cell membrane of leukocytes but sufficient to damage a part of the cell membrane; the labeling substance is one of being capable of passing through the damaged cell membrane and combining with an component contained in the leukocytes; and the treatment is performed at pH 3.0 to 11.0.

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for classifying and counting a specific cell in the field of clinical testing or cell study. More particularly, it relates to a method for labeling leukocytes contained in a blood sample. 2. Related Art Generally, in case of determining a specific cell contained in a biological sample containing a variety cells such as blood and urine, if the appearance of the cells (such as size and shape) is significantly different from each other, it is not difficult to distinguish one from others by using a conventional microscope of transmitted light type. For example, since erythrocytes, leukocytes and blood platelet contained in blood are different in size and shape with each other, they can be easily distinguished. In contrast, it is difficult to classify cells having a similar appearance with each other. For example, lymphocytes, monocytes, neutrophils, eosinophils and basophils which belong to subclasses of leukocytes are not easily classified. In general laboratories, components contained in cells such as a nucleus, granules, cytoplasm and endoenzyme in a leukocyte are stained with an appropriate dyeing solution to visualize the existence of the components contained in the cell and their amount and localization, thereby classifying and counting the cells. In other case, a device for measuring cell volume or a device for measuring a scattered light or fluorescent polarizing light of cell is utilized instead of using a microscope. Cells are detected by such a device by measuring the distinctive signals depending on the characteristics of the respective cell in order to classify the cells. If the distinctive signals are not obtained only by using such device, a suitable treatment is subjected to the cells so as to obtain available signals which are different among the cells. Examples of such treatments include a method for lysing cells other than a specific objective cell with a suitable cell lysing agent or a method for detecting the difference in volume of cells, or optical difference of scattered light, fluorescence light, absorbency, or the like. In order to detect the optical difference by using a scattered light, fluorescence light, absorbency and the like, it is preferred to combine a suitable labeling substance such as a dye with at least one component in the cell. It is known that components contained in cell are separated from outside by a membrane, called a cell membrane, such that the components are not leaked. However, the cell membrane does not work for complete sealing from outside. Rather, some substances necessary for survival of the cell are incorporated into the cell and waste products unnecessary for the cell are excreted from the cell. Thus, the cell membrane selects substances accurately and ingeniously to allow substances to pass through. Substances inside and outside the cell pass through various channels for ion passage provided in the membrane and pass through a lipid bilayer by dispersion. Such selective movement of substances continues as far as the cell is alive, but when the cell died, the selective operation is lost. In accordance with this phenomena, a method for distinguishing alive cells from dead cells is known as a dye-exclusion test, in which a dye which can invade into a damaged cell but can not invade into an undamaged cell is used as a labeling substance. An example of the well-known dye-exclusion test is to use a dye such as trypan blue and eosin. However, such a known method is not always carried out easily. That is, it is not possible to incorporate a labeling substance such as a dye into cells because cells naturally refuse the invasion of unnecessary substances to themselves, or even if it is possible it takes a long time. Generally, when cells are stained, various fixation is performed to the cells for inhibiting the invasion function or preventing deformation of cell and leak of components contained in the cell. In general, aldehydes such as formalin and glutaraldehyde, methanol and acetone are used as a fixing agent. However, the use of these fixing agents suffers from drawbacks such as involving a risk in handling and necessity of detoxication of waste water because of their toxicity which is not a low, requiring a long time, and a troublesome process for fixation. On the other hand, a method for generating optical difference without fixing is also known, in which selective substance exclusion function of cell membrane is inhibited to assist to combine the cell components with a dye which generally can not pass through the cell membrane. A well known example comprises the steps of lysing cell membrane and cytoplasm with a nonionic surfactant such as Triton X-100; staining the remaining nuclei with proridium iodide, ethidium bromide, etc. to prepare a test sample; followed by measuring a fluorescence light of the cell by a flow cytometer, microspectrophotometer, etc. and determining an amount of DNA. However, Triton X-100 lyses cell membranes and cytoplasms, and it also damage nuclei to no small extent, so that the data of DNA is made inaccurate. Accordingly, stabilization of nuclei, for example adding a stabilizer such as spermidine [N-(3-aminopropyl-1,4-butanediamine] or conducting any fixation is necessitated in this method. Moreover, as the nuclei are naked, it is not possible to precisely classify leukocytes into each subclass by using scattered light and the like. It is known to use two kinds of quaternary ammonium salt type surfactants for classifying leukocytes into two types i.e., mononucleocyte group and granulocyte group and analyzing them (for example disclosed in WO84/03771 and WO84/02777). However, the concentration of the surfactants is high, for example, 40 to 70 g/l for one quaternary ammonium salt type surfactant and 2 to 7 g/l for another, which destroys leukocytes themselves and nakes the nuclei, so that it was not possible to classify and counting leukocytes by measuring optical difference. Japanese Laid-Open Patent Application 88896/1986 discloses a method and a reagent for classifying and counting basophils which belong to leukocytes by using a water soluble surfactant in the limited pH range of 1.8 to 2.3 with a blood sample to generate optical difference to the basophils. However, the surfactant is also used at a high concentration of 10 to 20 g/l and the method fails to disclose the use of a labeling substance. Further, the European Patent Application 0 430 750 describes a method of treating a blood sample, in flow cytometry, with an aqueous solution comprising a cationic surfactant, such as a tertiary or quaternary ammonium salt at a concentration of 0,03 to 0,10 % (by volume), and a labeling substance (chlorazolblack), said solution having a pH of between 7,0 and 12,0. SUMMARY OF THE INVENTION The present invention provides a pretreatment method for blood analysis which comprises selective labeling of leukocytes by treating a blood sample with an aqueous solution comprising at least one surfactant selected from the group consisting of a cationic surfactant and an amphoteric surfactant, and with a labeling substance, characterized in that : the surfactant is contained in the aqueous solution at a concentration of 30 to 5,000 mg/l; the labeling substance is any substance which is capable of passing through the damaged cell membrane and combining with a component contained in leukocyte; and the treatment is performed at a pH of 3.0 to 11.0. According to the method of the present invention, only leukocytes can be selectively labeled in a simple manner by using untreated blood sample, and classified and counted by an optical means. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with lauryltrimethylammonium chloride as a surfactant. Fig. 2 is a histogram of the side scattered light when a blood sample is treated with lauryltrimethylammonium chloride as a surfactant. Fig. 3 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with lauryldimethylaminoacetic betaine as a surfactant. Fig. 4 is a histogram of the side scattered light when a blood sample is treated with lauryldimethylaminoacetic betaine as a surfactant. Fig. 5 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with benzyldimethylcetylammonium chloride as a surfactant. Fig. 6 is a histogram of the side scattered light when a blood sample is treated with benzyldimethylcetylammonium chloride as a surfactant. Fig. 7 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated without adding a nonionic surfactant. Fig. 8 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with C 16 H 33 O-(CH 2 CH 2 O) 10 -H as a nonionic surfactant. Fig. 9 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with C 16 H 33 O-(CH 2 CH 2 O) 20 -H as a nonionic surfactant. Fig. 10 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with C 16 H 33 O-(CH 2 CH 2 O) 30 -H as a nonionic surfactant. Fig. 11 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with C 12 H 25 O-(CH 2 CH 2 O) 30 -H as a nonionic surfactant. Fig. 12 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood is treated with C 18 H 37 O-(CH 2 CH 2 O) 20 -H as a nonionic surfactant. Fig. 13 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood is treated with C 18 H 35 O-(CH 2 CH 2 O) 20 -H as a nonionic surfactant. Fig. 14 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with C 18 H 37 COO-(CH 2 CH 2 O) 25 -H as a nonionic surfactant. Fig. 15 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with a nonionic surfactant having the following formula: Fig. 16 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with a nonionic surfactant having the following formula: Fig. 17 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with a cationic surfactant by adding ethanol (100 ml/l) as an aqueous alcohol. Fig. 18 is a histogram of the side scattered light when a blood sample is treated with a cationic surfactant by adding ethanol (100 ml/l) as an aqueous alcohol. Fig. 19 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the forward scattered light (FSC) when a blood sample is treated with a cationic surfactant by adding ethanol (100 ml/l) as an aqueous alcohol are referred to as the coordinated axes. Fig. 20 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with a cationic surfactant by adding ethanol (200 ml/l) as an aqueous alcohol. Fig. 21 is a histogram of the side scattered light when a blood sample is treated with a cationic surfactant by adding ethanol as an aqueous alcohol (200 ml/l). Fig. 22 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the forward scattered light (FSC) when a blood sample is treated with a cationic surfactant by adding ethanol (200 ml/l) as an aqueous alcohol. Fig. 23 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with a cationic surfactant by adding ethanol (400 ml/l) as an aqueous alcohol. Fig. 24 is a histogram of the side scattered light when a blood sample is treated with a cationic surfactant by adding ethanol (400 ml/l) as an aqueous alcohol. Fig. 25 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the forward scattered light (FSC) when a blood sample is treated with a cationic surfactant by adding ethanol (400 ml/l) as an aqueous alcohol. Fig. 26 is a scattergram showing the relationship between the intensity of the red fluorescence light (RFL) and the intensity of the green fluorescence light (GFL) when a venous blood sample of normal subjects is treated with the reagent composition of Example 4. Fig. 27 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a venous blood sample of normal subjects is treated with the reagent composition of Example 4. Fig. 28 is a scattergram showing the relationship between the intensity of the forward scattered light (FSC) and the intensity of the red fluorescence light (RFL) when a venous blood sample of normal subjects is treated with the reagent composition of Example 4. Fig. 29 is a scattergram showing the relationship between the intensity of the red fluorescence (RFL) and the intensity of the green fluorescence light (GFL) when a venous blood sample of a patient with acute myelocytic leukemia is treated with the reagent composition of Example 4. Fig. 30 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a venous blood sample of a patient with acute myelocytic leukemia is treated with the reagent composition of Example 4. Fig. 31 is a scattergram showing the relationship between the intensity of the forward scattered light (FSC) and the intensity of the red fluorescence light (RFL) when a venous blood sample of a patient with acute myelocytic leukemia is treated with the reagent composition of Example 4. Fig. 32 is a scattergram showing the relationship between the intensity of the red fluorescence light (RFL) and the intensity of the green fluorescence light (GFL) when subjects with atypical lymphocytes is treated with the reagent composition of Example 4. Fig. 33 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when subjects with atypical lymphocytes is treated with the reagent composition of Example 4. Fig. 34 is a scattergram showing the relationship between the intensity of the forward scattered light (FSC) and the intensity of the red fluorescence light (RFL) when subjects with atypical lymphocytes is treated with the reagent composition of Example 4. Fig. 35 is a scattergram showing the relationship between the intensity of the red fluorescence light (RFL) and the intensity of the green fluorescence light (GFL) when subjects with erythroblast and immature granulocytes is treated with the reagent composition of Example 4. Fig. 36 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when subjects with erythroblast and immature granulocytes is treated with the reagent composition of Example 4. Fig. 37 is a scattergram showing the relationship between the intensity of the forward scattered light (FSC) and the intensity of the red fluorescence light (RFL) when subjects with erythroblast and immature granulocytes is treated with the reagent composition of Example 4. Fig. 38 is a scattergram showing the relationship between the intensity of the red fluorescence light (RFL) and the intensity of the green fluorescence light (GFL) when a venous blood sample of normal subjects is treated with the reagent composition of Example 5. Fig. 39 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a venous blood sample of normal subjects is treated with the reagent composition of Example 5. Fig. 40 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and impedance signal (IMP) when a venous blood sample of normal subjects is treated with the reagent composition of Example 5. Fig. 41 is a histogram of the side scattered light when a venous blood sample of normal subjects is treated with the reagent composition of Example 5. Fig. 42 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the green fluorescence light (GFL) when a venous blood sample of normal subjects is treated with the reagent composition of Example 5. Fig. 43 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the forward scattered light (FSC) when a venous blood sample of normal subjects is treated with the reagent composition of Example 5. Fig. 44 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood sample is treated with the reagent composition of Reference Example 1. Fig. 45 is a histogram of the side scattered light when a blood sample is treated with the reagent composition of Reference Example 1. Fig. 46 is a scattergram showing the relationship between the intensity of the side scattered light (SSC) and the intensity of the red fluorescence light (RFL) when a blood is treated with the reagent composition of Reference Example 2. Fig. 47 is a histogram of the side scattered light when a blood sample is treated with the reagent composition of Reference Example 2. DETAILED DESCRIPTION OF THE INVENTION A blood sample used for the pretreatment method for blood analysis of the present invention means a sample derived from blood of human being and animals and may be bone marrow fluid. The blood sample must contain leukocytes. However, upon circumstances, the sample may be used after having removing erythrocytes and blood platelet. According to the method of the present invention, leukocytes can be selectively classified and counted by treating the whole blood sample without removing erythrocyte and blood platelet. The blood sample is usually treated with an anticoagulant before applying the method. In the present invention, an aqueous solution comprising at least one surfactant selected from the group consisting of a cationic surfactant and amphoteric surfactant. Preferably, the aqueous solution of the present invention contains the surfactant dissolved in an aqueous solvent, preferably water. Preferable examples of the cationic surfactants are quaternary ammonium salt type surfactant or pyridinium salt type surfactant. Specifically, the quaternary ammonium salt type surfactant can be represented by the formula (I): where R 1 , R 2 and R 3 are independently a hydrogen atom, a C 1-8 alkyl or a C 6-8 aralkyl group; R 4 is a C 8-18 alkyl group, a C 8-18 alkenyl or a C 6-18 aralkyl group; and X is an anion. Preferable examples of the C 1-8 all or C 6-8 aralkyl group represented by R 1 , R 2 and R 3 are methyl, ethyl, octyl, heptyl, hexyl and benzyl group, among which a C 1-3 alkyl group such as a methyl and ethyl group is more preferable. Preferable examples of the C 8-18 alkyl group, C 8-18 alkenyl group or C 6-18 aralkyl group represented by R 4 are octyl, decyl, dodecyl, tetradecyl and benzyl group. The pyridinium salt type surfactant can be represented by the formula (II): where R 5 is a C 8-18 all group and X is an anion group. Preferable examples of the C 8-18 alkyl group represented by R 5 are a C 10-18 straight chain all group such as decyl, dodecyl and tetradecyl group. Specific examples of these cationic surfactants are lauryltrimethylammonium chloride, myristyltrimethylammonium chloride, cetyltrimethylammonium chloride, cetyldimethylethylammonium chloride and benzyldimethylcetylammonium chloride. The amphoteric surfactant includes a betaine type surfactant having the formula (III): where R 1 and R 2 are independently a hydrogen atom, a C 1-8 alkyl or a C 6-8 aralkyl group; R 4 is a C 8-18 alkyl group, a C 8-18 alkenyl group and a C 6-18 aralkyl group; and n is 1 or 2. Specific examples of the betaine type surfactant include lauryldimethylaminoacetic betaine and stearyldimethylaminoacetic betaine. In the present invention, the aqueous solution comprising the cationic or amphoteric surfactant is used at an concentration not to destroy the whole cell membrane of leukocytes but sufficient to damage a part of the cell membrane. The surfactant is used at a concentration of in the range of about 30 to 5,000 mg/l, preferably about 50 to 2,000 mg/l, more preferably about 50 to 1,500 mg/l. If the surfactant is used in an excess amount, labeling function of some labeling substances is inhibited by the surfactants. Also, the surfactant contained at a higher concentration than the above upper limit acts on cells and make nuclei naked, so that optical difference is hardly measured by using a scattered light. The aqueous surfactant solution is preferably used in 2-200 by volume per one volume of the blood sample. It is not recommended to dilute the blood sample too much with the surfactant solution because components of blood other than leukocytes, for example erythrocytes, are not lysed, thereby causing a difficulty of the measurement. The labeling substance usable in the present invention is any substance that can pass through damaged cell membrane of leukocyte and be combined with components contained therein. For example, a fluorescence dye is preferably used when a flow cytometer for measuring a general fluorescence light and scattered light is used. In case of using a flow cytometer for measuring absorbency, a dye other than fluorescence dye can be used. Alternatively, a substance which can generate a dye by reacting with cell components is also preferably used. Such labeling substances are known in publications related to a flow cytometer. In this specification, the phrase "combined with cell components" means ionic bonding between the cell components and labeling substance or covalent bonding between proteins (amino group) contained in the cell and the labeling substance. As examples of the labeling substances that can be combined with the cell nucleus (DNA), ethidium bromide (EB) and propidium iodide (PI) are well known. In addition, most of basic dyes can be used for staining nuclei. Examples of the labeling substances that can be combined with RNA include Pyronin Y, Acridine Orange, Tiazole Orange, Acridine Orange 10 dodecyl bromide and Auramine-O. Alternative examples of the labeling substances that can react with proteins in the cell (amino group) include fluorescein isothiocyanate (FITC) and 7-chloro-4-nitrobenzoxazole (NBD-CI). As described above, dyes other than those generally used for a flow cytometer can be used. For example, a dye generally used for measuring electric potential of cell membrane such as 3,3'-dihexyloxacarbocyanine (DiOC6 (3)) can be also used because such a dye passes through a cell membrane so easily that it can stain components contained in the cell such as granules. According to the present invention, a distinctive stain is exhibited by staining the treated cells, which is different from the stain exhibited by staining normal alive cells or fixed cells. Therefore, the present invention can be also used in order to develop usage of dyes which have been used for staining cells for the purpose other than classifying and counting leukocytes, or to examine a possibility of usage of dyes which have not been used for staining cells. The labeling substance is added at a concentration of 1 to 500 mg/l, preferably about 1 to 200 mg/l. As an applied example of the present invention, nucleate and anucleate cells are distinguished by treating the cationic or amphoteric surfactant, followed by staining with the labeling substance capable of combining cell nucleus (DNA), for example, DNA probes such as propidium iodide and ethidium bromide. In addition, in case where a flow cytometer capable of measuring fluorescence or scattered light is used, fluorescent basic dyes are suitably used as the labeling substance. In the present invention, when the surfactant and labeling substance are acted on the blood sample, the pH range of the solution is preferably adjusted to pH 3.0-11.0, more preferably from pH 4.0 to 11.0. In case of detecting eosinophils by using side scattered light, it is preferable to be at pH 5.0-11.0. Further, if the formation of a bonding between the labeling substance and cell components or the acting of the surfactant on cells depends on pH value, a suitable buffer may be added for maintaining desired pH. The method for treating blood sample with the surfactant and the labeling substance is not specifically limited, but is conducted by a known method. For example, an aqueous reagent solution comprising the surfactant and the labeling substance is mixed with the blood sample which is pretreated with an anticoagulant. In this case, the pH of the aqueous reagent solution may be adjusted to the above range by adding a buffer. The buffer may be added simultaneously to the reaction system. The method of the present invention can be generally carried out at a temperature from a room temperature to slightly elevated temperature, for example about 10 to 50°C. The reaction according to the method of the present invention generally proceeds very fast, for example completes in 5 to 30 seconds. Therefore, it is found that the present invention is suitably adapted for classifying and counting leukocytes by a high speed automatic analysis device. In the present invention, a nonionic surfactant may be added to the aqueous reagent solution of the cationic or amphoteric surfactant and the labeling substance. Preferably, the nonionic surfactant comprises at least one selected from polyoxyethylene glycol type agents having the formula: R 6 -R 7 -(CH 2 CH 2 O) n -H wherein R 6 is a C 8-22 alkyl group or a C 8-22 alkenyl group, R 7 is -O-, -COO-, or and n is an integer of 10 or more. The nonionic surfactant of this type is economically advantageous because it is available at a low price. Among them, the nonionic surfactant of n (addition molar number) being 10 or more are particularly preferable because of water-solubility and less cytotoxicity. Specific examples of the polyoxyethyleneglycol surfactants are 20 molar addition product of polyoxyethyleneglycol nonylphenyl ether, 30 molar addition product of polyoxyethyleneglycol cetyl ether, and 20 molar addition product of polyoxyethyleneglycol oleyl ether. Further, it is possible to use other nonionic surfactants than the above mentioned polyoxyethyleneglycol type, e.g., those having HLB (hydrophile-liophile balance) value of 13 or more such as Tween type (polyoxyethylene sorbitan alkylate). The amount of the above nonionic surfactant is preferred to be used at a concentration of 100 to 10,000 mg/l, more preferably, at a concentration of 100 to 5,000 mg/l, which varies depending upon the kind of the cationic or amphoteric surfactant and other conditions. The mechanism of the function of the nonionic surfactant is not made clear, but is considered that the nonionic surfactant is combined with the surface of cell to control the acting of the ionic surfactant toward cell membrane, whereby inhibiting the lysis of cell components with the ionic surfactants. For example, the use of the ionic surfactant associated with the nonionic surfactant is preferable when the ionic surfactant has so potent lysing activity in an extent that leukocytes are unnecessarily damaged. The nonionic surfactant also will act to accelerate the lysis of erythrocytes, which causes a problem in case of measuring leukocytes contained in blood sample. Further, it can act to solubilize substances which are precipitated from a reaction solution by neutralization of anionic substances included in cell components or others of the blood sample, with the cationic surfactant. In addition, it can exert to inhibit aggregation of erythrocytes which remain as a ghost without their complete lysing, which will occur in case where erythrocytes are rich as in a test sample of blood product and the dilution of said test sample with the aqueous reagent solution is obliged to be low. In such case where the dilution ratio of the aqueous reagent solution must be low, the nonionic surfactant is effectively used otherwise it is not necessary. Alternatively, the nonionic surfactant generally shows a solubility for effect for a water-insoluble labeling substance, which is the merit of increasing the number of the available labeling substances. According to the method of the present invention, a water soluble alcohol can be further contained in the aqueous reagent solution when the blood sample is treated with the aqueous reagent solution of at least one cationic or amphoteric surfactant. Examples of the water soluble alcohols include a water soluble aliphatic alcohol having 2 to 5 carbon atoms such as ethyl alcohol, propyl alcohol, isopropyl alcohol and t-butyl alcohol, among which a branched chain alcohol such as isopropyl alcohol or t-butyl alcohol is preferable. The similar effect can be exhibited by using an alkoxyalcohol such as methoxyethanol or an aromatic alcohol such as phenethyl alcohol. The concentration of the water soluble alcohol to be added depends on the type of the cationic or amphoteric surfactant or other conditions. In case of using ethyl alcohol or isopropyl alcohol, it is preferably used at a concentration of 50 to 400 ml/l or 25 to 200 ml/l, with its half concentration of each alcohol having one extra carbon atom. The water soluble alcohol acts to selectively enhance the function of the ionic surfactant, thereby damaging cell membranes at a lower concentration of the ionic surfactant. Also, it denatures proteins contained in cell and makes them to be insoluble. Accordingly, the use of the water soluble alcohol is effective to minimize the loss of cytoplasms and granules and optimize the damage of the cell membrane. Therefore, the water soluble alcohol also acts for maintaining the effect of obtaining optical difference measured by scattered light and the like. Further, the water soluble alcohol has an effect to facilitate the lysis of erythrocytes. In using the blood sample subjected to the method of the present invention, the labeled cells can be easily classified and counted, for example, by an optical means such as flow cytometer or the like, or other known means. From another aspect of the present invention, it provides a blood analysis reagent suitable for applying the method of the present invention. The preferred reagent used for the present invention is as follows: (a) a reagent comprising: about 50 to 5,000 mg/l of a cationic or amphoteric surfactant, about 1 to 200 mg/l of labeling substance, and an aqueous medium of water or water soluble alcohol to be balanced to one liter. (b) a reagent comprising: about 50 to 5,000 mg/l of a cationic or amphoteric surfactant , about 100 to 10,000 mg/l of a nonionic surfactant, about 1 to 200 mg/l a labeling substance, and an aqueous medium of water, or water and water soluble alcohol to be balanced to one liter. (c) a reagent of (a) and (b) above, in which a buffer is contained to adjust pH to pH 3.0-11.0. The function of the ingredients of the reagent composition of the present invention is further described as follows. The surfactant of the present invention is believed to function to draw a part of substances which constitutes a cell membrane, probably lipid molecules, thereby yielding pores in cell membrane which can pass a substance which does not usually pass the membrane, that is the damage of cell membrane. The effect of the present invention can be achieved by this function. In particular, a labeling substance such as a dye is allowed to come into a cell and rapidly combine with an intracellular component. For example, in case that the combination is formed by an ionic bond as set forth below, the labeling completes at almost instantly. As a secondary effect, the cationic and amphoteric surfactant having a positive charge in its molecule have a function that the positive charge is ionically bound with the intracellular components having negative charge (for example, RNA with a phosphoric group, protein with a carboxyl group, and the like), whereby the intra-cellular components becomes to be insoluble. The insolubilized components do not leak out from the cell even when its membrane is damaged. Moreover, the cell which accumulates the insolubilized components is prevented in leaking of most of cytoplasm, nucleus, and granules of cells. Therefore, optical difference can be unexpectedly obtained by measuring the side scattered light as described hereinafter, when the reagent is suitably prepared. EXAMPLES Example 1: Concentration of cationic or amphoteric surfactant An aqueous solution having the following composition: HEPES-NaOH buffer (PH 7.0) 10 mM Labeling substance (Ethidium bromide (EB)) 50 mg/l was mixed with an aqueous solution (1 ml) comprising various surfactant and venous blood (25 µl) which was treated with an anticoagulant. The resulting solution was put into a flow cytomerter where red fluorescence light and side scattered light were measured to determine the concentration of the surfactant at which staining with EB is available. The results are shown in Table 1. Surface Active Agent Concentration Lauryltrimethylammonium chloride (LTAC) 500 mg/l Mrystyltrimethylammonium chloride 100 mg/l Cetyltrimethylammonium chloride 50 mg/l Cetylpyridinium chloride 50 mg/l Lauryldimethylaminoacetic betaine (Anon BL, produced by NIHON YUSHI Co.) 500 mg/l Stearyldimethylaminoacetic betaine 500 mg/l Cetyldimethylethylammonium bromide 50 mg/l Benzyldimethylcetylammonium chloride 50 mg/l As shown in Table 1, the stronger hydrophobicity of surfactant has, the lower concentration the agent is used at, the weeker the surfactant has, the higher concentration the agent is used at. Figure 1 shows a scattergram by the side scattered light (SSC) and red fluorescence light (RFL) obtained by using lauryltrimethylammonium chloride (hereinafter referred to LTAC) as the surfactant are referred to as the coordinated axes. In Figure 1, leukocytes can be distinguished from other blood corpuscles by the difference of red fluorescence light signals. Further, as is clearly shown from Figure 1, lymphocytes Ly; leukocytes Othl other than lymphocytes Ly and eosinophils Eo; and eosinophils Eo can be classified and counted. Fig. 2 is a histogram of the side scattered light obtained by the same experiment. Similarly, lymphocytes, granulocyte Othl other than lymphocytes and eosinophils, and eosinophils can be classified and counted from Figure 2. Figures 3 and 4 are a scattergram and histogram in case of using lauryldimethylaminoacetic betaine (Anon BL) as the surfactant. In this case, lymphocytes Ly; leukocytes Othl other than lymphocytes Ly and eosinophils Eo; and eosinophils Eo can be also classified and counted. Other surfactants having a strong lysing property classify leukocytes into 2 groups under the condition described above. For example, Figures 5 and 6 show the case when benzyldimethylcetylammonium chloride was used as the surfactant. It is seen from these figures that leukocytes were classified and counted only into two groups for eosinophils Eo and other leukocytes Oth2. The lysing property of the surfactants can be adjusted by adding a nonionic surfactant, thereby obtaining more detailed classification as described later. Thus, leukocytes can be unexpectedly classified by using a reagent comprising the ionic surfactant that has been considered not to be available for generating optical difference. Upon observing the relationship the surfactant and the performance of leukocytes classification, it was found that the extent of cell damage was increased in accordance with the increase of the hydrophobicity of the surfactant, resulting in the difficulty of the classification. In addition, it was observed that the classification was made difficult by the increase of the concentration of the surfactants. Example 2: Effect of containing the nonionic surfactant A reagent having the following composition was prepared. HEPES-NaOH buffer (PH 7.0) 10 mM Ethidium bromide (EB) 50 mg/l LTAC 1,000 mg/l Nonionic surfactant 1,000 mg/l To the reagent (1.0 ml) having the above composition, venous blood (25 µl) which was treated with an anticoagulant was mixed. The red fluorescence light and side scattered light were measured by a flow cytometer. The results are shown in Figures 7 to 16. Figure 7 shows a scattergram wherein the side scattered light (SSC) and red fluorescence light (RFL) obtained without using an ionic surfactant are referred to as the coordinated axes. In this case, the lysing is strengthened by increasing the amount of LTAC, thereby giving an excessive damage to the leukocytes. Unlike the results seen from Figure 1 (LTAC 500 mg/l), it was observed that leukocytes are aggregated into one, so that the leukocytes can not be classified. In contrast, Figures 8 to 16 show that when various nonionic surfactants were contained in the surfactant, the damage of leukocytes was controlled and leukocytes can be classified and counted into lymphocytes Ly, eosinophils Eo, and other leukocytes Othl with respect to every nonionic surfactant that was used. The molar number of addition of polyoxyethylene glycol and type of hydrophobic group did not affect the results. Example 3: Function of water soluble alcohol A reagent having the following composition was prepared. HEPES-NaOH buffer (PH 7.0) 10 mM Ethidium bromide (EB) 50 mg/l LTAC 250 mg/l nonionic surfactant C 16 H 33 O(CH 2 CH 2 O) 30 -H 1,000 mg/l Ethanol 100-400 ml/l To the reagent (1.0 ml) having the above composition, venous blood (25 µl) which was treated with an anticoagulant was mixed. The red fluorescence light, forward scattered light, and side scattered light were measured by a flow cytometer. Figures 17 to 19 show a scattergram wherein the side scattered light (SSC) and red fluorescence light (RFL) obtained when ethanol (100 ml/l) was contained are referred to as the coordinated axes; a histogram thereof; and a scattergram obtained in the same manner as above by using the side scattered light (SSC) and red fluorescence light (RFL). As seen from these figures, leukocytes are classified and counted into four groups, i.e., lymphocytes Ly, monocytes Mo, eosinophils Eo, and other leukocytes Oth3. Figures 20 to 22 show the case that ethanol (200 ml/l) was contained. In this case, leukocytes are also classified and counted into four groups, i.e., lymphocytes Ly, monocytes Mo, eosinophils Eo, and other leukocytes Oth3. Figures 23 to 25 show the case that ethanol (400 ml/l) was contained. In this case, leukocytes are classified and counted into three groups and counted, i.e., lymphocytes Ly, eosinophils Eo, and other leukocytes Oth1. Accordingly, it is observed that excessive use of ethanol prevents from obtaining optical difference of cells. It is also observed that the lysis of erythrocytes is facilitated in accordance with the increase of the number of carbon atoms. Example 4: Reagent compositions Example A reagent having the following composition were prepared. Lauryltrimethylammonium chloride (LTAC) (cationic surfactant) 1 g Brij35 (polyoxyethyleneglycol lauryl ether/ molar number of addition: 23) (nonionic surfactant) 1 g Acridine Orange 10-dodecyl bromide (AO-10) (labeling substance: dye) 0.100 g Ethyl alcohol 0.100 g Citric acid mono hydrate 2.1 g NaOH adequate amount Purified water 0.9 liter To the reagent (1.0 ml) having the above composition, venous blood (25 µl) which was treated with an anticoagulant was mixed. After 20 seconds at a room temperature, the red fluorescence light, green fluorescence light, forward scattered light, and side scattered light were measured by a flow cytometer. Figures 26 to 28 show scattergrams obtained as results of normal subjects wherein the red fluorescence light (RFL) and green fluorescence light (GFL), side scattered light (SSC) and red fluorescence light (RFL), and forward scatlight (FSC) and red fluorescence light (RFL) are respectively referred to as the coordinated axes. As seen from each figure, leukocytes are classified into three groups, i.e., lymphocytes Ly, monocytes Mo and granulocytes Gr. Incidentally, De indicates reduced erythrocyte membrane and blood platelet. LTAC used in the present invention is a cationic surfactant and has a property of: i) giving a damage to erythrocyte membranes to leak hemoglobins contained therein to convert the erythrocytes into a ghost, thereby making the erythrocytes optically transparent, and ii) giving a damage to leukocyte membranes so as to allow dye AO-10 to pass through the cell membranes. The mechanism of the function of LTAC for damaging membrane of erythrocyte and leukocyte is not clear. However, it is considered that a part of lipid which constitutes cell membrane is lysed by the surface active action, resulting in forming trace pores in the cell membrane that allows to pass hemoglobin and dye. Brij 35 is a nonionic surfactant. It controls the action of LTAC to inhibit from giving a severe damage to the degree that nuclei are made naked or the leukocytes are deformed. The agent also has a function for controlling the aggregation of erythocytes that are converted into ghosts. Further, Brij 35 works for making AO-10 soluble, which is hard to be dissolved in water. The dye AO-10 has a dodecyl group at 10th position of Acridine Orange which is often used for staining DNA and RNA simultaneously. Since AO-10 does not usually pass through a cell membrane because of the steric hindrance of the long chain alkyl group, it merely stains cell membranes and the components contained in cells can not be stained. Therefore, the dye has been used only as a hydrophobic probe. According to the present invention, as a cell membrane is damaged, AO-10 can invade into the cell and combine with components contained therein. When the dye is used for staining a blood as shown in Examples, it combines with various components in the cell in a specific manner. For example, in cytoplasm of lymphocytes and monocytes, AO-10 combines with RNA present in the cytoplasm by an ionic bond and stains the components orange, while a green fluorescence light is emitted from nucleus and granules thereof. It is inferred that in accordance with a conventionally known function of hydrophobic probe, the long chain alkyl group of AO-10 is invaded through the nucleus membranes and granulocyte membrane to combine with the nucleus and granulocyte. In case of neutrophils, since the cytoplasm rarely has RNA, a green fluorescence light is emitted. From nucleus and granulocyte of neutrophils, a green light fluorescence light is emitted similar to the case from nucleus and granulocyte of the lymphocytes and monocytes. The reason for these phenomena are considered that metachromatism occurred when AO-10 was combined with RNA, thereby staining RNA orange, while metachromasy did not occur in nucleus and granulocyte because AO-10 was combined with the surface of nucleus and granulocyte, thereby emitting a green fluorescence light. LTAC is combined with RNA by an ionic bond and the electric charges are neutralized, so that RNA are made not to be lost. It is considered that AO-10 is combined with such components which are made not to be lysed. In Example of the present invention, a blood sample sampled from patients having a disease in blood were measured by using the reagent composition of the present invention. Figures 29 to 31 show the results obtained from the samples sampled from patients having an acute myelocytic leukemia. Myeloblasts My were classified and counted. Figures 32 to 34 show the results obtained from the samples in which Atypical lymphocytes Aly are erupted. Atypical lymphocytes Aly were classified and counted. Figures 35 to 37 show the results obtained from the samples in which erythroblasts Er and immature granulocytes Im were erupted. As seen from these figures, Erythroblasts Er and immature granulocytes Im were classified and counted. Since Blast cells Er and My, Atypical lymphocytes Aly, Immature granulocytes Im include RNA richly therein, they can be detected by staining RNA. In addition, Reticlocytes Ret were partially detected from Figure 37. In this example, Erythrocytes are not completely lysed, but rather, only the membrane of Erythrocyte was damaged. Therefore, cell membranes of the cells which are converted into a ghost and RNA of the cell components are stained, thereby detecting Reticlocytes. Conventionally, dyes have not been used for the purpose of the present invention because dyes are hard to pass through cell membranes. However, according to the present invention, an effect of dye which is different from the conventional use can be exhibited. Example 5: Reagent Composition Example A reagent having the following composition were prepared. Acridine Orange 10-dodecyl bromide (AO-10) (label substance) 0.010 g DiOC6 (3) (label substance) 0.020 g Lauryltrimethylammonium chloride (LTAC) (cationic surfactant) 0.5 g Polyoxyethyleneglycol cetyl ether (molar number of addition: about 40) (nonionic surfactant) 1.0 g Isopropyl alcohol 0.050 liter NaCl 4.0 g HEPES 2.3 g NaOH Adequate Amount Purified water 0.95 liter To the reagent (1.0 ml) having the above composition, venous blood (25 µl) of normal subjects which was treated with an anticoagulant was mixed. After 20 seconds at a room temperature, the red fluorescence light, green fluorescence light, forward scattered light, side scattered light, and impedance signal were measured by a complex flow cytometer available for detecting not only optical difference but also impedance signals (IMP) based on the change of electric resistance. In the present example, the damage given to leukocytes is made minimum by using a smaller amount of cationic surfactants compared with the amount used in Example 4. In addition, as the reagent composition includes NaCl, impedance signals can be measured, and as the composition has pH 5.0 or more, eosinophils can be also measured. Further, the dye DiOC6 (3) capable for staining granules well was added for facilitating separation of granules when measured by the red fluorescence light and green fluorescence light. Figures 38 to 43 show scattergrams and a histogram when using the above regent composition wherein the red fluorescence light (RFL) and green fluorescence light (GFL) (Fig.38), side scattered light (SSC) and red fluorescence light (RFL) (Fig.39), and side scattered light (SSC) and impedance signals (IMP) (Fig.40) are respectively referred to as the coordinated axes; side scattered light histogram (Fig.41); scattergrams wherein side scattered light (SSC) and green fluorescence light (GFL) (Fig.42), and side scattered light (SSC) and forward fluorescence light (FSC) (Fig.43) are respectively referred to as the coordinated axes. In these figures, Ne+Ba denotes a distribution of neutrophils and basophils. In the present example, similar to Example 4, RNA can be stained. Therefore, Blast cells Er and My, Atypical lymphocytes Aly, Immature granulocytes Im can be classified and counted. Incidentally, a flow cytometer disclosed in Japanese Patent Application Laid-Open H5-34251 (1993) (USSN 07/902,979) was used for in this example to detect impedance signals. Example 6: Reagent composition example A reagent having the following composition were prepared. Lauryldimethylaminoacetic betaine (amphoteric surfactant) (Anon BL: produced by NIHON YUSHI Co.) 2 g Polyoxyethylene glycol nonylphenyl ether (molar number of addition: 20) (nonionic surfactant) 1 g Isopropyl alcohol 0.100 liter Citric acid mono hydrate 2.1 g NaOH Adequate Amount Propidium iodide 0.1 g Purified water 0.9 liter To the reagent (800 µl) having the above composition, venous blood (400 µl) which was treated with an anticoagulant was combined, and side scattered light and red fluorescence light were measured by a flow cytometer. The present example aims to detect a trace amount of leukocytes in a reduced dilution which remain after the treatment by a filter for eliminating leukocytes. Generally, lysing agent available for obtaining volume differences is used in 100 to 200 times diluted concentration, so that it was not possible to use it for the preparation of the reagent composition having a high concentration such as those of the present example. In case of using Triton X-100 conventionally used for a blood preparation, the ratio of blood:lysing agent is 1:7.5. However, more concentrated reagent composition for measurement can be prepared in the present invention. Further, according to the present invention, a scatter-gram similar to Figures 3 and 4 can be obtained and leukocytes can be classified into 3 groups. It is not possible if Triton X-100 is used. Thus, reagent compositions having higher concentration at which the measurement is not possible by conventional methods can be used by the method of the present invention. Reference Example 1 For comparison, a reagent described in the specification of WO 84/003771 (a reagent comprising a cationic surfactant at a high concentration) was prepared as follows: Dodecyltrimethylammonium chloride (cationic surfactant) 55 g/l Tetradecyltrimethyltrimethylammonium chloride (Cationic surfactant) 9 g/l Cyanic pottasium 0.300 g/l Polyoxyethylene glycol alkylphenol ether (Nonionic surfactant) 12 ml/l To the reagent (1.0 ml) having the above composition, venous blood (25 µl) which was treated with an anticoagulant was combined. After incubating for 20 seconds, the side scattered light and red fluorescence light were measured by a flow cytometer. The results are shown in Figures 44 and 45. As seen from the figures, the leukocytes are aggregated into one population, so that they can not be classified by the side scattered light and red fluorescence light based on the optical difference. Reference Example 2 A reagent which comprises a nonionic surfactant was prepared as follows and performed a DNA staining. Nonidet P-40 (Nonionic surfactant) 1 ml/l Sodium Citrate 10 g/l To the reagent (1.0 ml) having the above composition, propidium iodide (50 µg/ml) was added, then venous blood (25 µl) which was treated with an anticoagulant was combined. After incubating for 20 seconds, the side scattered light and red fluorescence light were measured by a flow cytometer. The results are shown in Figures 46 and 47. As seen from the figures, the eosinophils can be identified by the side scattered light and red fluorescence light based on the optical difference because they were not naked. However, leukocytes other than eosinophils can not be classified into subclasses. According to the present invention, 1. the property of labeling substance for passing through cell membrane can be enhanced by treating a biological sample with an aqueous solution comprising the cationic and/or amphoteric surfactant and with the labeling substance without a treatment such as fixing. As a result, cells can be labeled with various labeling substances almost instantly. In addition, while eliminating the influence of erythrocytes, optical difference can be obtained by the scattered light and the like by using the surfactants which have not been available. 2. Since the problem of transmittance of labeling substance to cell membrane is solved, labeling substances which have been considered not to be used for cell classification can be made available for such classification. 3. As the results, a property of labeling substances different from the conventional property can be found (for example, portion available to dye, etc.). 4. Precise classification which has not been achieved in prior art can be made by using the nonionic surfactant and alcohol. 5. The present invention can be also used for examining the property of labeling substances.

Claims: A pretreatment method for blood analysis which comprises treating a blood sample with an aqueous solution comprising at least one surfactant selected from the group consisting of a cationic surfactant and an amphoteric surfactant, and with at least one labeling substance to selectively label leukocytes, characterized in that: the surfactant is contained in the aqueous solution at a concentration of 30 to 5,000mg/l; the labeling substance is one of being capable of passing through the damaged cell membrane and combining with a component contained in the leukocyte; and the treatment is performed at pH 3.0 to 11.0. The pretreatment method for blood analysis according to claim 1, wherein the surfactant is contained at a concentration of 50 to 2,000 mg/l. The pretreatment method for blood analysis according to claim 1, wherein the cationic surfactant is a quaternary ammonium salt type surfactant or pyridinium salt type surfactant. The pretreatment method for blood analysis according to claim 3, wherein the quaternary ammonium salt type surfactant has the formula (I) : where R 1 , R 2 and R 3 are independently a hydrogen atom, a C 1-8 alkyl group or a C 6-8 aralkyl group; R 4 is a C 8-18 alkyl group, a C 8-18 alkenyl group or a C 6-18 aralkyl group, and X is an anion. The pretreatment method for blood analysis according to claim 3, wherein the quaternary ammonium salt type surfactant is at least one selected from the group consisting of : lauryltrimethylammonium chloride, myristyltrimethylammonium chloride, cetyltrimethylammonium chloride, cetyldimethylethylammonium chloride, and benzyldimethylcetylammonium chloride. The pretreatment method for blood analysis according to claim 3, wherein the pyridinium salt type surfactant has the formula (II) : where R 5 is a C 8-18 alkyl group and X is an anion. The pretreatment method for blood analysis according to claim 6, wherein the pyridinium salt type surfactant is cetylpyridinium chloride. The pretreatment method for blood analysis according to claim 1, wherein the amphoteric surfactant has the formula (III) : where R 1 and R 2 are independently a hydrogen atom, a C 1-8 alkyl group or a C 6-8 aralkyl group; R 4 is a C 8-18 alkyl group, a C 8-18 alkenyl group or a C 8-18 aralkyl group; and n is 1 or 2. The pretreatment method for blood analysis according to claim 8, wherein the amphoteric surfactant is lauryldimethylaminoacetic betaine or stearyldimethylaminoacetic betaine. The pretreatment method for blood analysis according to claim 1, wherein the aqueous solution of the surfactant is used at 2 to 200 by volume per one volume of blood sample. The pretreatment method for blood analysis according to claim 1, wherein a nonionic surfactant is added to the aqueous solution of the surfactant at a concentration of 100 to 10,000 mg/l. The pretreatment method for blood analysis according to claim 11, wherein the nonionic surfactant is contained at a concentration of 100 to 5,000 mg/l. The pretreatment method for blood analysis according to claim 1, wherein a water-soluble alcohol is added in the aqueous solution of the surfactant. The pretreatment method for blood analysis according to claim 13, wherein the water-soluble alcohol is ethyl alcohol and used at a concentration of 50 to 400 ml/l. The pretreatment method for blood analysis according to claim 13, wherein the water-soluble alcohol is isopropyl alcohol and used at a concentration of 25 to 200 ml/l. The pretreatment method for blood analysis according to claim 1, wherein the labeling substance is a dye capable of combining with a nucleus of leukocyte. The pretreatment method for blood analysis according to claim 16, wherein the labeling substance is ethidium bromide or propidium iodide. The pretreatment method for blood analysis according to claim 1, wherein the labeling substance is a dye capable of combining with an RNA contained in a leukocyte. The pretreatment method for blood analysis according to claim 18, wherein the labeling substance is Pyronin Y, Acridine Orange, Tiazole Orange, Acridine Orange 10 dodecyl bromide or Auramine-O. The pretreatment method for blood analysis according to claim 1, wherein the labeling substance is added at a concentration of 1 to 500 mg to one liter of the aqueous solution of the surfactant. The pretreatment method for blood analysis according to claim 1, wherein the labeled leukocytes are classified and counted by an optical means. The pretreatment method for blood analysis according to claim 1, wherein the treatment is performed at pH 4.0 to 9.0.

Inventors: SAKATA TAKASHI, SAKATA, TAKASHI
Applicants: SYSMEX CORP, SYSMEX CORPORATION
Application year: 1993
Main classification: G01N1
IPC codes: G01N33, G01N15