Reagent useful for detection and quantitative determination of leukocytes in biological fluids and method of using it

Abstract: The invention concerns a reagent for the detection and quantitative determination of leukocytes by measuring the myelo­ peroxidase (MPO) activity of biological samples, which is enough sensitive for disclosing even only a few leukocytes, without inter­ ferences caused by hemoglobin even in the presence of several ery­ throcytes, and suitable for photometric readings in the visible spectrum region. The reagent comprises a buffer, a chromogen, a surface-active agent, at least an alkali metal halide, a hydro­ peroxide compound and optionally a reaction promoter. The invention also relates to a method of using the reagent by splitting it into two component parts.

Description: The present invention refers to a reagent for the detection and quantitative determination of leukocytes in samples of human and animal body fluids, and to a method of using it. The detection of leukocytes in body fluids, such as, e.g., urine, is very useful in the diagnosis of some types of infections, e.g. in the uro-genital tract. For the detection of leukocytes in the urine two different methods are at present employed: the microscopic examination and the determination of particular enzyme activities which can be correlated with the presence of leukocytes themselves. The microscopic examination of sample represents the classical method for detecting leukocytes and is considered as the reference method in that it allows the objective control of the presence of the cells (leukocytes) in the test sample, directly or after enrichment by centrifugation. However, such a method, besides being too laborious for extensive utilization in analysis laboratories, does not allow the identification of leukocyte cells having undergone lysis caused by excessive alkalinity of sample or a too wide time interval elapsed from the taking of the sample and its microscopic examination, which represents a serious drawback. For strongly alkaline samples, as it often happens in case of infection of urinary ducts, cell lysis can reach completion even within one hour. Under such circumstances, the risk of considering as negative samples, which are on the contrary positive, is very high. Esterases are the enzymes at present employed for the indirect detection of leukocytes, in that they split particular synthetic substrates by hydrolysis delivering groups which spontaneously become coloured by air oxidation or after reaction with other appropriate substances. This method is the one typically employed in reactive stripes which contain the reagents dried on inert supports and are used for detection of leukocytes in urine. The reaction stripes containing the dried reagents to be dipped in the test sample allow a semi-quantitative determination of leukocyte esterase activity, which requires a time period of at least 60-120 seconds per sample. The detection of leukocytes carried out through the measurement of peroxidase enzyme activity is also known from a long time. In U.S. Patent 3.087.794 a method is described, which is useful for distinguishing the peroxidase activity of leukocytes from that of erythrocytes. The utilization of hydrogen peroxide (or another peroxide) at different concentrations in the two cases permits such a differentiation: o-tolidine, which is a cancerous substance, is the employed chromogen. The method described in the above-mentioned U.S. Patent, however, does not reach the sensitivity levels which are at present requested and is not even able to distinguish leukocytes from erythrocytes with certainty, if the concentration of these latter is much higher than that of the former. The leukocytes typically present in urine in case of renal and uro-genital infections are neutrophilic granulocytes. They have the typical microbicidal function correlated to the phagocytosis, that is carried out through different mechanisms, not yet perfectly understood. Among them, the most important one appears to be the microbicidal and detoxicating MPO-H₂O₂ -halide system. With MPO it is hereinafter meant the leukocyte myeloperoxidase enzyme. MPO is an enzyme present in large amount in polymorphonuclear leukocytes (neutrophils granulocytes), representing up to 5% of the whole cell mass. The determination of MPO activity can therefore allow the detection of the presence of the above mentioned leukocytes in biological samples or in human or animal body fluids. When the halide compound in MPO-H₂O₂-halide system is iodide (I⁻), the following reactions can take place: J. Bos et al. (Infect. Immun. 1981, 32, 427) exploy just the reactions (a) and (b) for determining the myeloperoxidase activity of neutrophils and the peroxidase activity of eosinophils in blood, by carrying out spectrophotometric readings at 360 nm of the reaction product (I₃). However, the UV spectrophotometric reading involves many practical drawbacks and brings about many non-specificities when applied to samples having extremely variable compositions, such as urine. Methods are also known for determining the MPO activity in blood, which are comparable to the ones already extensively employed for the peroxidase of vegetable origin (HRPO) and employ o-dianisidine, o-tolidine or guaiacol as acceptor-chromogens. These methods too are however non-specific because they do not distinguish among peroxidase activity, MPO activity and hemoglobin pseudo-peroxidase activity. It has now been surprisingly found a reagent suitable both for the detection and for the quantitative determination of leukocytes in biological samples through the measurement of MPO activity, enough sensitive for detecting even only a few leukocytes, having such a specificity that hemoglobin does not bring about interferences at all even in the presence of several erythrocytes and being suitable for photometric readings in the visible spectrum region. The reagent according to the present invention comprises a MPO specific substrate, such as an alkali metal halide compound; 4-aminoantipyrine as chromogen; a buffer for maintaining the optimum desired pH value; a surface-active agent for aiding and accelerating the lysis of leukocytes in order to release MPO making it immediately available for the reactions of interest; a hydroperoxide compound, and optionally a promoter for increasing the system sensitivity. More particularly, such reagent comprises as components at least a buffer selected from the group consisting of citrate, succinate, acetate, and formate in concentration of 2.5-500 mmol/l; a chromogen represented by 4-aminoantipyrine in concentration of 5-1000 mmol/l; at least a surface-active agent selected from the group formed by non-ionic and anionic surface-active agents, in concentration of 0.05-2% ; at least an alkali metal halide in concentration of 2-1000 mmol/l; and at least a hydroperoxide compound in concentration of 2-100 mmol/l. Preferably the buffer is in concentration of 2.5-50 mmol/l; the chromogen is in a concentration of 200-500 mmol/l; the surface-active agent is in concentration of about 0.2%; the alkali metal halide is in a concentration of 10-20 mmol/l; and the hydroperoxide compound is selected from the group consisting of hydrogen peroxide, in a concentration of about 5-25 mmol/l, and magnesium monoperoxyphtalate, in a concentration of about 40-60 mmol/l. Hydroperoxide compounds other than hydrogen peroxide and magnesium monoperoxyphtalate can be used, such as urea hydrogen peroxide, both alone and in mixture with each other in the reagent. Substances such as MPOS (3-(N-morpholino)propane-sulfonic acid), imidazole and/or 8-aminoquinoline, in concentrations from 10 - 300 mmol/l can be present, for increasing the reagent sensitivity. Expecially 8-aminoquinoline can bring about a clear promoting effect, preferably in concentration of 30-40 mmol/l, so as to increase reaction sensitivity. The reaction involved by the chromogen activity is considered to be as follows: The reaction rate which can be achieved for the detection and quantitative determination of leukocytes allows the examination of the sample in about 30 seconds when the components are used in the above specified optimum amounts; if said components are used in amounts which are not the optimum ones but falling within the indicated ranges, an effective detection and quantitative determination of the leukocytes is still effectively possible even if the operating time is longer. For the practical utilization of reagent and method according to the present invention the essential thing is a conventional filter photometer for readings in the visible spectrum region, even if the utilization of more complex and sophisticated instruments, particularly suitable for automation and acceleration of analytical procedures cannot be excluded. The reagent of the present invention can be employed when its components are included in only one or even in two component parts. When the reagent is used in a single component part containing all the above-mentioned components, it is necessary to do the detection and quantitative determination of leukocytes quickly with precision because the components immediately react with the biological substrate; the components of the reagent can also be split into two component parts which are, one after the other, reacted with the biological fluids. A component part can in fact include the alkali metal halide and the other part the hydroperoxide compound, the remaining components being included in one or both component parts, except for the chromogen being only in the component part that is first added to the biological fluid sample. The chromogen 4-aminoantipyrine allows a coloured oxidized compound to be formed, its colour intensity being such as to allow the determination of MPO activity by means of spectrophotometric readings in the visible spectrum region (520-540 nm) with sufficient sensitivity and specificity. The alkali metal halide, preferably potassium iodide, is used as MPO specific substrate in the reagent. The buffers preferably used are citrate (because it gives more stability to the reagent) or formate (because it increases reaction sensitivity). In order to accelerate the leukocyte lysis for releasing MPO and making it immediately available for the reaction of interest, 0.1-2%, preferably about 0.2%, of an anionic surface-active agent, such as the sodium salts of secondary alkyl sulfonates (Teepol 610 R ) or a nonionic surface-active agent, such as polyoxyethylene(20)sorbitan monolaurate (Tween 20 R ), polyoxyethylene(20)sorbitan monoleate (Tween 80 R ), polyoxyethylene(23)lauryl ether (Brij 35 R ), higher alcohol polyoxyethylene ethers (Triton x-67 R ; Triton x-100 R ; Triton x-305 R ), preferably polyethylene glycol p-isooctylphenyl ether (Triton x-100 R ) can suitably be added to the reagent. The following Examples are illustrative, but not limitative of the present invention. EXAMPLE 1 A reagent is prepared formed by a first component part, consisting of citrate buffer (196 mmol/l, pH 5.0) containing 4-aminoantipyrine (62 mmol/l), potassium iodide (130 mmol/l), 8-aminoquinoline (28 mmol/l) and Triton x-100 R (10 g/l); and by a second component part, consisting of hydrogen peroxide (20 mmol/l) in distilled water. In a spectrophotometry cuvette, thermoregulated at 37°C and having 1 cm of optical path, are successively mixed: 2.0 ml of distilled water and 0.5 ml of a fresh urine sample containing varied amounts of leukocytes, previously measured by microscopic counting of the sediment obtained by centrifugation of the sample. After addition of 0.5 ml of the first component part, the absorbance (A₁) at 540 nm is measured and then 0.1 ml of the second component part is added. The resulting mixture has pH 5.0. Exactly 30 seconds after the addition of the second component part, the absorbance (A₂) at 540 nm is measured. The calculated absorbance variation (A₂-A₁) obtained in 30 seconds is then correlated with the leukocyte content of the sample. The obtained results are summarized in Table I. Leukocytes per µl (A₂-A₁) 0 0.036 0 0.042 0 0.028 0 0.040 25 0.070 25 0.070 25-30 (28) 0.078 75 0.085 75-80 (78) 0.092 500 0.702 700 0.908 Leukocytes per µl: number of leukocytes per µl of fresh urine, measured by microscopic counting of the sediment obtained by centrifugation of the sample. (A₂-A₁): Absorbance variation obtained in 30 seconds. EXAMPLE 2 The same procedure of Example 1 is repeated, with the exception that the reagent consists of a first component part, containing succinate buffer (20 mmol/l, pH 7.2), 4-aminoantipyrine (62 mmol/l), potassium iodide (130 mmol/l), 8-aminoquinoline (28 mmol/l) and Triton x-100 R (20 g/l); and of a second component part, containing hydrogen peroxide (20 mmol/l) in citrate buffer (100 mmol/l, pH 4.7). The resulting mixture has the same pH 5.0 as the Example 1, notwithstanding the different pH of the compositions. The calculated absorbance variation (A₂-A₁) obtained in 30 seconds is correlated with the leukocyte content of the sample. The obtained results are summarized in Table II. Leukocytes per µl (A₂-A₁) 0 0.040 0 0.044 0 0.032 0 0.040 25 0.081 25 0.078 25-30 (28) 0.078 75 0.092 75-80 (78) 0.093 500 0.775 700 1.010 Leukocytes per µl: number of leukocytes per µl of fresh urine, measured by microscopic counting of the sediment obtained by centrifugation of the sample. (A₁-A₁): Abosrbance variation obtained in 30 seconds. ESEMPIO 3 In a spectrophotometry cuvette, thermoregulated at 37°C and having 1 cm of optical path, are successively mixed: 2.0 ml of distilled water and 0.5 ml of a fresh urine sample composed by a mixture of a group of negative urines or by a mixture of a group of positive urines containing 200 leukocytes/µl, determined by microscopic counting in the sediment obtained by centrifugation of the sample. After addition of 0.5 ml of the reagent first component part consisting of acetate buffer (100 mmol/l, pH 4.8) containing 4-aminoantipyrine (40 mmol/l), 8-aminoquinoline (25 mmol/l), Triton X-100 R (15 g/l) and potassium iodide in concentrations ranging between 100 and 175 mmol/l, the absorbance (A₁) at 530 nm is measured; and then 0.1 ml of the second component part, consisting of hydrogen peroxide (20 mmol/l), is added. Exactly 30 seconds after the addition, the absorbance (A₂) at 530 nm is measured. The calculated absorbance variation (A₂-A₁) in 30 seconds is correlated with the iodide concentration. The obtained results are summarized in Table III. Iodide mmol/l N P (P-N) in the reagent in the cuvette 100 16.12 0.032 0.211 0.179 125 20.16 0.048 0.320 0.272 150 24.80 0.098 0.388 0.290 175 28.22 0.148 0.387 0.239 N = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of negative urines P = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of positive urines (P-N) = difference between the (A₂-A₁) values of the positive sample and those of the negative sample. EXAMPLE 4 The same procedure of Example 3 is repeated, with the exception that, in this case the reagent comprises a first component part, consisting of acetate buffer (100 mmol/l, pH 4.8) containing 4-aminoantipyrine (40 mmol/l), 8-aminoquinoline (25 mmol/l), Triton X-305 R (20 g/l) and hydrogen peroxide in concentrations ranging from 5 to 7 mmol/l; and a second component part, consisting of potassium iodide (600 mmol/l), and the following results are obtained, as reported in Table IV. Peroxide mmol/l N P (P-N) in the Reagent in the cuvette 5 0.81 0.048 0.410 0.362 6 0.96 0.105 0.460 0.355 7 1.13 0.180 0.500 0.320 N = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of negative urines P = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of positive urines (P-N) = difference between the (A₂-A₁) values of the positive sample and those of the negative sample. EXAMPLE 5 The same procedure of Example 3 is repeated, whith the exception that, in this case, the reagent comprises a first component part, consisting of acetate buffer (100 mmol/l), 8-aminoquinoline (25 mmol/l), potassium iodide (140 mmol/l), Triton X-67 R (2.5 g/l) and 4-aminoantipyrine in concentrations ranging from 40 to 320 mmol/l; and a second component part, consisting of hydrogen peroxide (25 mmol/l) in citrate buffer (100 mmol/l, pH 4.8). The employed samples are formed by a mixture of a group of negative urines and by a mixture of a group of positive urines containing 70 and 250 leukocytes/µl, measured by microscopic counting of the sediment obtained by centrifugation of the sample. The obtained results are summarized in Table V. 4-aminoantipirine mmol/l N P70 P250 In the reagent in the cuvette 40 6.45 0.048 0.142 0.488 80 12.90 0.060 0.145 0.512 120 19.35 0.058 0.140 0.470 160 25.81 0.056 0.148 0.465 320 51.61 0.045 0.150 0.413 N = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of negative urines P70 = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of positive urines containing 70 leukocytes/µl P250 = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of positive urines containing 250 leukocytes/µl EXAMPLE 6 In a spectrophotometry cuvette, having 1 cm of optical path and thermoregulated at 37°C, are successively mixed: 2.0 ml of distilled water and 0.5 ml of a fresh urine sample formed by a mixture of a group of negative urines or by a mixture of a group of positive urines containing 250 leukocytes/µl, measured by microscopic counting of the sediment obtained by centrifugation of the sample. After the addition of 0.5 ml of a first component part of the reagent, consisting of acetate buffer (100 mmol/l, pH 4.8) containing 4-aminoantipyrine (40 mmol/l), potassium iodide (140 mmol/l), Tween-20 R (10 g/l) and 8-aminoquinoline in concentrations ranging from 15 to 40 mmol/l, the absorbance (A₁) at 530 nm is measured; and then 0.1 ml of a second component part of the reagent, consisting of magnesium monoperoxyphtalate (50 mmol/l), is added. The calculated absorbance variation (A₂-A₁) in 30 seconds is correlated with the concentration of 8-aminoquinoline. The obtained results are summarized in Table VI 8-Aminoquinoline mmol/l N P (P-N) in the reagent in the vessel 15 2.42 0.041 0.405 0.360 20 3.23 0.045 0.460 0.415 30 4.84 0.047 0.458 0.411 40 6.45 0.051 0.461 0.410 N = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of negative urines P = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of positive urines (P-N) = difference between the (A₂-A₁) values of the positive sample and those of the negative sample. EXAMPLE 7 In a spectrophotometry cuvette, having 1 cm of optical path and thermoregulated at 37°C, are mixed successively 2.0 ml of distilled water and 0.5 ml of a fresh urine sample containing varied amounts of leukocytes, measured by microscopic counting of the sediment obtained by centrifugation of the sample. After the addition of 0.5 ml of the first component part of the reagent, consisting of citrate buffer (175 mmol/l, pH 4.8) containing 4-aminoantipyrine (50 mmol/l), potassium iodide (120 mmol/l) and Tween-80 R (10 g/l), the absorbance (A₁) at 540 nm is measured in two series of tests carried out in the presence or not of 8-aminoquinoline as promoter (31 mmol/l) in the first component part. Then, 0.1 ml of a second component part of the reagent, consisting of hydrogen peroxide (25 mmol/l) in citrate buffer (100 mmol/l, pH 4.8), is added. Exactly 30 seconds after the addition, the absorbance (A₂) at 540 nm is measured. The calculated absorbance variation (A₂-A₁) in 30 seconds is correlated with the leukocyte content of the sample sediment; the obtained results are summarized in Table VII. Leukocytes per µl (A₂-A₁) Reagent without promoter Reagent with promoter 0 (Negative) 0.025 0.040 25 0.050 0.085 50 0.065 0.110 100 0.150 0.223 200 0.220 0.370 300 0.345 0.580 (A₂-A₁): absorbance variation in 30 seconds Leukocytes per µl = Number of leukocytes per µl of fresh urine measured by microscopic counting of the sediment obtained by centrifugation of the sample. EXAMPLE 8 In a spectrophotometry cuvette having 1 cm of optical path and thermoregulated at 37°C, are mixed successively: 2.0 ml of distilled water and 0.5 ml of a urine sample formed by a mixture of a group of negative urines or by a mixture of a group of positive urines containing 250 leukocytes/µl, measured by microscopic counting of the sediment obtained by centrifugation of the sample. After addition of 0.5 ml of the first component part of the reagent, consisting of citrate buffer (100 mmol/l) at pH ranging from 4.0 t 7.0 and containing 4-aminoantipyrine (40 mmol/l), potassium iodide (140 mmol/l), 8-aminoquinoline (25 mmol/l) and Triton X-100 R (15 g/l), the absorbance (A₁) at 530 nm is measured; 0.1 ml of a second component part of the reagent, consisting of hydrogen peroxide (25 mmol/l) in distilled water, is then added and, exactly 30 seconds after the addition of this latter, the absorbance (A₂) at 530 nm is measured. The obtained results are summarized in Table VIII pH (A₂-A₁) N P (P-N) 4.0 0.050 0.061 0.011 4.5 0.037 0.400 0.363 4.8 0.045 0.415 0.370 5.0 0.044 0.408 0.364 5.5 0.047 0.368 0.321 6.0 0.038 0.288 0.250 6.5 0.010 0.120 0.110 7.0 0.012 0.012 0.000 N = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of negative urines P = (A₂-A₁) at 530 nm per 0.5 ml of a sample of the group of positive urines (P-N) = difference between the (A₂-A₁) values of the positive sample and those of the negative sample. EXAMPLE 9 In a spectrophotometry cuvette having 1 cm of optical path and thermoregulated at 37°C, are mixed successively 1.6 ml of distilled water, 0.5 ml of fresh urine sample containing leukocytes or erythrocytes in known varied amounts, determined by microscopic counting of the sediment obtained by centrifugation of the sample, and 0.5 ml of the first component part of the reagent, consisting of citrate buffer (20 mmol/l, pH 7.2) containing 4-aminoantipyrine (62 mmol/l), potassium iodide (132 mmol/l) and Brij-35 R (2 g/l). The absorbance (A₁) at 530 nm is measured and then 0.5 ml of a second component part of the reagent, containing citrate buffer (200 mmol/l, pH 4.4), hydrogen peroxide (6.0 mmol/l) and 8-amino-quinoline (30 mmol/l) are added. Exactly after 30 seconds after the addition of second component part, the absorbance (A₂) at 530 nm is mesured. The calculated absorbance variation (A₂-A₁) is correlated with the leukocyte or erythrocyte content of the sample. The obtained results are summarized in Table IX. Leukocytes/µl Erythrocytes/µl (A₂-A₁) 0 0 0.035 0 0 0.044 15 0 0.058 20 0 0.068 35 0 0.102 250 0 0.399 0 10 0.038 0 50 0.036 0 250 0.042 0 500 0.040 (A₂-A₁): Absorbance variation after 30 seconds. EXAMPLE 10 In a spectrophotometry cuvette, having 1 cm of optical path and thermoregulated at 37°C, are mixed successively 1.6 ml of distilled water, 0.5 ml of a fresh urine sample containing leukocytes and erythrocytes in known varied amounts, determined by microscopic counting of the sediment obtained by centrifugation of the sample, and 0.5 ml of the first component part of the reagent, consisting of formate buffer (20 mmol/l, pH 7.1) containing 4-aminoantipyrine (120 mmol/l), potassium iodide (130 mmol/l) and Triton x-100 R (2 g/l) ; the absorbance (A₁) at 530 nm is measured and then 0.1 ml of a second component part of the reagent, consisting of a buffer (300 mmol/l pH 4.4) of a compound selected from citrate, formate succinate or acetate, containing hydrogen peroxide (5.7 mmol/l) and 8-aminoquinoline (31 mmol/l) are added. Exactly 30 seconds after the addition of the second component part, the absorbance (A₂) at 530 nm is measured. The calculated absorbance variation (A₂-A₁) is correlated with the leukocyte or erythrocyte content of the sample. The obtained results are summarized in Table X Leukocytes/µl Erythrocytes/µl (A₂-A₁) Reagent with citrate Reagent with formate Reagent with succinate Reagent with acetate 0 0 0.034 0.048 0.046 0.039 0 0 0.038 0.049 0.044 0.037 15 0 0.059 0.074 0.068 0.062 25 0 0.081 0.107 0.090 0.085 75 0 0.164 0.210 0.187 0.190 500 0 0.872 1.204 0.920 0.898 0 10 0.032 0.061 0.059 0.042 0 50 0.041 0.055 0.058 0.038 0 250 0.039 0.057 0.054 0.038 0 500 0.044 0.057 0.060 0.039 0 1000 0.052 0.066 0.062 0.051 (A₂-A₁): Absorbance variation after 30 seconds. EXAMPLE 11 In a spectrophotometry cuvette, having 1 cm of optical path and thermoregulated at 37°C, are mixed successively: 2.0 ml of distilled water, 0.5 ml of a fresh urine sample formed by a mixture of a group of negative urines or by a mixture of a group of positive urines containing 250 leukocytes/µl, measured by microscopic counting of the sediment obtained by centrifugation of the sample, and 0.5 ml of the reagent consisting of succinate buffer (100 mmol/l, pH 4.8) containing 4-aminoantipyrine (123 mmol/l), potassium iodide (132 mmol/l), 8-aminoquinoline (31 mmol/l); hydrogen peroxide (6 mmol/l) and varied surface - active agents (3.0 g/l). The absorbance at 520 nm is measured immediately (A₁) and exactly after seconds (A₂). In table XI are summarized the obtained results. Surface-active agent (A₂-A₁) N P (P-N) None 0.033 0.092 0.059 Teepol 610 R 0.018 0.088 0.070 Litium dodecyl sulfate 0.008 0.118 0.110 Sodium dodecyl sulfate 0.011 0.099 0.088 Sodium dioctylsulphosuccinate 0.011 0.069 0.058 Sodium pentanesulfonate 0.019 0.110 0.091 Sodium hexanesulfonate 0.018 0.106 0.088 Brij 35 R 0.032 0.420 0.388 Triton x-67 R 0.048 0.502 0.454 Triton x-100 R 0.041 0.496 0.455 Triton x-305 R 0.037 0.377 0.340 Tween 20 R 0.035 0.501 0.466 Tween 80 R 0.023 0.491 0.468 N = (A₂-A₁) at 520 nm per 0.5 ml of a sample of the group of negative urines. P = (A₂-A₁) at 520 nm per 0.5 ml of a sample of the group of positive urines. (P-N) = difference between the (A₂-A₁) values of the positive sample and those of the negative sample. EXAMPLE 12 In a spectrophotometric cuvette, having 1 cm of optical path and thermoregulated at 37°C, are mixed successively: 2.0 ml of distilled water, 0.8 ml of a fresh urine sample containing varied amounts of leukocytes, previously measured by microscopic counting of the sediment obtained by centrifugation of the sample, and 0.5 ml of the first component part of the reagent, consisting of citrate buffer (5 mmol/l, ph 8.0) containing 4-aminoantipyrine (400 mmol/l), potassium iodide (14.5 mmol/l) and Triton x-100 R (2g/l) in distilled water; after the addition of 0.5 ml of the second component part of the reagent, consisting of citrate buffer (266 mmol/l, ph 4.4), hydrogen peroxide (18 mmol/l), and 8-aminoquinoline (31 mmol/l) in dimethylsulfoxide (112 ml/l) in distilled water, the variation of absorbance (A₂-A₁) is measured at 546 nm for 20 sec. The calculated absorbance variation (A₂-A₁) is correlated with the leukocyte contents of the sample. The obtained results are summarized in Table XII LEUKOCYTES per µl (A₂-A₁) 0 0.038 0 0.036 0 0.027 10 - 15 0.045 20 0.049 25 0.055 25 - 30 0.059 70 - 80 0.082 100 - 130 0.110 600 0.398 900 0.588 (A₂-A₁): Absorbance variation for 20 seconds.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, GR, IT, LI, LU, NL, SE A reagent for the detection and quantitative determination of leukocytes in biological fluids by photometric reading in the visible spectrum region, comprising as components at least a buffer selected from the group consisting of citrate, succinate, acetate, and formate in a concentration of 2.5-500 mmol/l; 4-aminoantipyrine as chromogen in a concentration of 5-1000 mmol/l; at least a surface-active agent, selected from the group formed by non-ionic and anionic surface-active agents, in a concentration of 0.05-2% ; at least an alkali metal halide in a concentration of 2-1000 mmol/l; and at least a hydroperoxide compound in a concentration of 2-100 mmol/l. The reagent according to claim 1, characterized in that said hydroperoxide compound is selected from the group consisting of hydrogen peroxide, magnesium monoperoxyphtalate and urea hydrogen peroxide. The reagent according to claims 1-2, characterized in that the buffer is in a concentration of 2.5-50 mmol/l, the chromogen is in a concentration of 200-500 mmol/l; the surface-active agent is in a concentration of about 0.2%; the alkali metal halide is in a concentration of 10-20 mmol/l; the hydrogen peroxide is in a concentration of 5-25 mmol/l and the magnesium monoperoxyphtalate is in a concentration of 40-60 mmol/l. The reagent according to claims 1-3, characterized in that the surface-active agent is a higher alcohol polyoxyethylene ether, preferably polyethylene glycol p-isooctylphenyl ether. The reagent according to claims 1-4, characterized in that it comprises also at least a promoter selected from the group formed by 3-(N-morpholino)-propanesulfonic acid, 8-aminoquinoline and imidazole, in a concentration of 10-300 mmol/l. The reagent according to claim 5, characterized in that the promoter is 8-aminoquinoline in a concentration of 30-40 mmol/l. The reagent according to claims 1-6, characterized in that said buffer is citrate or formate. The reagent according to claims 1-7, characterized in that said alkali metal halide is potassium iodide. A method of using the reagent according to claims 1-8, characterized in that the components of said reagent are split into two component parts, one of which includes the alkali metal halide while the other includes the hydroperoxide compound, and the remaining components are included in one or both component parts, except for the chromogen which is present only in the one of the two component parts that is first added to the biological fluids. Claims for the following Contracting State : ES A method for the detection and quantitative determination of leukocytes in biological fluids, characterized in that a reagent comprising as components at least a buffer selected from the group consisting of citrate, succinate, acetate, and formate in a concentration of 25-500 mmol/l, 4-aminoantipyrine as chromogen in a concentration of 5-1000 mmol/l; at least a surface-active agent, selected from the group formed by non-ionic and anionic surface-active agents, in a concentration of 0.05-2% ; at least an alkali metal halide in a concentration of 2-1000 mmol/l; and at least a hydroperoxide compound in a concentration of 2-100 mmol/l, is added to a biological fluid, and a photometric reading in the visible spectrum region is effected for detecting and determining the amount of leukocytes in said fluid. A method according to claim 1, characterized in that said reagent, contains as component the hydroperoxide compound selected from the group consisting of hydrogen peroxide, magnesium monoperoxyphtalate and urea hydrogen peroxide. A method according to claims 1-2, characterized in that said reagent according to claims 1-2, contains as components the buffer in a concentration of 2.5-50 mmol/l, the chromogen in a concentration of 200-500 mmol/l; the surface-active agent in a concentration of about 0,2%; the alkali metal halide in a concentration of 10-20 mmol/l; the hydrogen peroxide in a concentration of about 5-25 mmol/l and the magnesium monoperoxyphtalate in a concentration of about 40-60 mmol/l. A method according to claims 1-3, characterized in that said reagent contains as component a surface-active agent selected from higher alcohol polyoxyethylene ether, preferably polyethylene glycol p-isoctylphenyl ether. A method according to claims 1-4, characterized in that said reagent comprises also as component at least a promoter selected from the group formed by 3-(N-morpholino)-propanesulfonic acid, 8-aminoquinoline and imidazole, in a concentration of 10-300 mmol/l. A method according to claims 1-5, characterized in that said reagent comprise as promoter 8-aminoquinoline in a concentration of 30-40 mmol/l. A method according to claims 1-6, characterized in that said reagent comprises as component a buffer selected from citrate or formate. A method according to claims 1-7, characterized in that said reagent comprises potassium iodide as said alkali metal halide. A method of using the reagent employed in the method according to claims 1-8,characterized in that the components of said reagent are split into two component parts, one of which includes the alkali metal halide while the other includes the hydroperoxide compound, and the remaining components are included in one or both component parts, except for the chromogen which is present only in the one of the two component parts that is first added to the biological fluids.

Inventors: MEIATTINI FRANCO, MEIATTINI, FRANCO
Applicants: DIESSE DIAGNOSTICA, DIESSE DIAGNOSTICA SENESE S.R.L.
Application year: 1990
Main classification: C12Q1
IPC codes: C12Q1