JP3522865B2 - Urine leukocyte detection method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting leukocytes in urine, and more particularly to a method for detecting leukocytes in urine which can be detected including lysed leukocytes by measuring the concentration of myeloperoxidase in urine.

[0002]

Urine leukocytes are an important index in urinary tract infections and inflammatory diseases of the urinary tract. Conventionally, urinary leukocytes are exclusively examined by urinary sediment microscopy and test strip methods (esterase activity). Has been measured. This test strip method uses, for example, 3- (N-toluenesulfonyl-L-alaniryloxy) -5-phenylpyrrole as a substrate, and 3-hydroxy-5-one which is one of the hydrolysates by neutrophil esterase. Phenylpyrrole is subjected to diazo coupling with 1-diazo-2-naphthol-4-sulfonic acid, and the reaction of the substance produced by the reaction to give a purple color is visually compared to examine leukocyte urine.

[0003]

However, according to the study by the present inventor, there are not a few false-negative cases presumed to be due to leukolysis in the sediment microscopy and test strip methods. It was suggested. That is, 95% of white blood cells in urine
Is a neutrophil, the urinary neutrophil-derived protein concentration in daily samples was compared with the measurement results by urinary sediment microscopy and test strip methods, and the results in Figs. 1 and 2 were obtained. The
The presence of false negative cases was suggested. As a neutrophil-derived protein, lactoferrin (Lf) present in special granules
Attention was paid to myeloperoxidase (MPO) present in azure granules. In FIG. 1, the urinary sediment white blood cell count was microscopically examined by preparing a sample according to the method for preparing a urinary sediment sample defined by the Japan Society for Clinical Hygiene Inspection. In addition, in the measurement of FIG. 2 (and later), the urine test strip is AM company, B
The one manufactured by M Co. was used. On the other hand, the urinary neutrophil-derived protein in FIGS. 1 and 2 was measured by an ELISA method using an anti-human polyclonal antibody. In particular,
(1) 50 μl of a urine sample containing a surfactant (Triton X100) at a final concentration of 0.05% is sampled in the well of a Mypro plate on which polyclonal antibodies against each protein are fixed. (2) After incubating at 37 ℃ for 1 hour,
Wash with pure water and add 2.5 μl / well to the well of the microplate.
Alkaline phosphatase (AL
P) Dispense 100 μl of the labeled antibody. (3) Incubate at 37 ℃ for 1 hour, wash with pure water, and then KIND
Dispense 100 μl of KING method ALP substrate. (4) 3
After incubating at 7 ° C. for 30 minutes, 100 μl of the ALP color development solution was dispensed and photometry was performed at 510/630 nm to calculate the concentration of each protein (lactoferrin, myeloperoxidase). As is clear from FIGS. 1 and 2, MPO and L
There were discordant cases showing high f values, suggesting the presence of false negative cases due to leukolysis in both sedimentoscopy and test strip methods. The present invention has been made in view of this problem, and an object of the present invention is to provide a urine leukocyte detection method capable of detecting urine leukocytes more reliably.

[0004]

In order to achieve the above object, in the present invention, leukocytes in urine are detected by measuring the concentration of myeloperoxidase in urine. Also,
By measuring the urinary myeloperoxidase concentration and the urinary lactoferrin concentration and using the low urine myeloperoxidase concentration and the high urinary lactoferrin concentration alone as indicators, it is possible to detect nephritis in the early stage. it can. Hereinafter, the effects of the present invention will be described while describing the contents of the study until the completion of the present invention.

[Problems of urinary leukocyte count method (sedimentoscopy method, counting method)] Based on the results of FIGS. 1 and 2, the present inventor firstly disrupts white blood cells in urine. We examined the actual situation. FIG. 3 is a diagram showing the distribution of leukocyte disintegration rates for urine samples of 75 daily samples. Specifically, 30 leukocytes were added to the supernatant obtained by removing the cell components by centrifugation.
The disintegration rate (%) was obtained by adding 00 to 4000 cells / μl, allowing to stand at 37 ° C. for 15 hours, and measuring with a Coulter STKS (automatic blood cell counter). In addition, about 60 neutrophils
%, Lymphocytes of about 40% were used, but the average value of the disintegration rate was 33.3% (minimum 0.0%, maximum 93.8%). Conventionally, it has been pointed out that urine osmotic pressure and urine pH are related to the breakdown of leukocytes in urine. Next, the urine osmotic pressure and urine pH of the above-mentioned daily samples were measured. In addition, the urine osmotic pressure used the urine osmotic pressure measuring device (OM-6010) of Kyoto Daiichi Scientific. The relationship between the urine osmotic pressure and the urine pH and the leukocyte disintegration rate is as shown in FIGS. 4 (a) and 4 (b). Although there is no special relationship between the urine osmotic pressure and the leukocyte disintegration rate, The middle leukocyte disintegration rate was about 20% when the urine pH was 5 to 6, and it was revealed that the disintegration rate rapidly increases as the alkalinity increases.

Next, in order to identify the factors involved in leukocyte lysis, the relationship between the standing time and leukocyte lysis rate, the relationship between standing temperature and leukocyte lysis rate, and the relationship between pH and leukocyte lysis rate were investigated.
FIG. 5 (a) shows the time course of the disintegration rate when white blood cells were added to a urine sample having a pH of 7.5 as in the case of FIG. 3 and left at 37 ° C. for 25 hours. Figure 5
(B) is a comparison of the leukolysis rate when a certain urine sample was divided into 3 minutes and leukocytes were added in the same manner as in FIG. 3 and then left at 4 ° C., 25 ° C. and 37 ° C. for 15 hours. Is.
Further, FIG. 5 (c) shows that the same urine sample (pool urine) was adjusted to pH 3 with HCl or NaOH.
It is a diagram showing the difference in the leukolysis rate after standing for 15 hours at 7 ° C. The results shown in FIG. 5 suggest that alkaline urine having a pH of about 9 is particularly strongly disintegrated, and that enzymes are involved in the disintegration of leukocytes in urine. However, even about 25% of acid urine is disintegrated.

Therefore, next, 0.15 mo having different pH values is used.
leukocytes were added to each of the phosphate buffers of 1 to 3000-40
Add 00 units / μl, and then add 50 units / ml of acid protease or alkaline protease at 15 ℃ at 37 ℃.
After allowing to stand for a period of time, the difference in leukocyte disintegration rate depending on the pH value was examined (FIG. 6 (a)). In addition, 0.15mo with different pH values
White blood cells 3000-4000 / μ in 1 l phosphate buffer
1 was added, and the mixture was allowed to stand at 37 ° C. for 15 hours, and leukolysis caused by autolysis was examined (FIG. 6 (b)). From the results of FIGS. 6 (a) and 6 (b), it is considered that the leukocyte disintegration on the acidic side of urine pH is mainly due to the acid protease derived from bacteria.
Leukocyte disintegration on the H-alkaline side is considered to be due to autolysis by protease in leukocytes and the action of bacterial alkaline protease.

FIG. 7 (a) shows that urine sample supernatant from which cell components have been removed, to which 3000 to 4000 leukocytes / μl have been added, and another inhibitor has been added, respectively, at 37 ° C. for 15 hours. The urinary leukocyte collapse inhibition rate was calculated by obtaining the urine leukocyte count. The inhibitor added is EDTA (10 mm
ol), ABSF (1 mmol), EDTA (10 mm
ol) + ABSF (1 mmol), and ABSF is a serine protease inhibitor [4- (2-Amin
oethyl) benzensulfonyl Fluoride Hydrochloride]. It is considered that the urinary leukocyte destruction was prevented by the enzyme inhibitor, as shown in FIG. 7 (a), because the urinary leukolysis phenomenon was mainly due to the action of the enzyme. In particular, the combined use of serine protease inhibitor and EDTA which is a metalloprotease inhibitor was effective.

FIG. 7 (b) shows that the pH value is adjusted to pH 5-9.
Leukocytes were added to 15 to 30 mol of phosphate buffer solution for 3000 to 400
Inhibitors were added to 0 cells / μl and left at 37 ° C. for 15 hours, and leukocyte disintegration due to autolysis in the absence of bacteria was examined. As an inhibitor, ED
TA (10 mmol), ABSF (1 mmol), α1
-The inhibition rate of leukocyte autolysis was determined using antitrypsin (300 mg / dl). According to the results of FIG. 7 (b), α1-antitrypsin as an inhibitor against serine protease in leukocytes is ineffective in inhibiting leukocyte autolysis, and a low-molecular-weight serine protease inhibitor is significantly effective. Became clear. From the above,
Inhibitors that can act by entering leukocytes are considered to be effective in preventing leukocyte breakdown due to autolysis.
Since urinary leukocytes are destroyed by the action of an enzyme before urination, it is considered that urinary tract infections and urinary tract inflammatory diseases may be overlooked by simply counting the number of urinary leukocytes.

[Problems of Test Paper Method (Enzyme Activity Measurement Method)]
Next, the discordant urine shown in FIG. 2, which was negative in the test strip method but positive in the neutrophil-derived protein, was further examined. The supernatant from which cell components were removed by centrifugation was added with 500 leukocytes / μ
When the test paper method was carried out by adding l, the above-mentioned inconsistent urine (NO.1 to NO.5) showed various levels of esterase activity as compared with the control in which 500 leukocytes / μl were added to normal urine. Inhibition was observed (Fig. 8). Therefore, in order to estimate the molecular weight of urinary components that inhibit esterase activity,
Dialysis of inconsistent urine against physiological saline revealed that the esterase inhibitor was a polymer.
Then, gel filtration (Sephacryl 100) was performed, and 500 leukocytes / μl were added to each fraction and the esterase activity was measured by the test paper method. The results shown in FIG. 9 were obtained. As shown in FIG. 9, the presence of an esterase activity inhibitor was observed in the fraction having a molecular weight of about 60,000.

When blood cells were added with 500 leukocytes / μl, the test paper method revealed no reaction (Fig. 10).
(E)), since the serum reacts somewhat with dilution to 2-fold or 4-fold (FIGS. 10 (f) and (g)), the inhibitor for the test strip method is also present in serum. Is confirmed. It is known that proteases that hydrolyze amino acid or peptide esters such as trypsin have esterase activity, and therefore involvement of trypsin inhibitors as inhibitors is considered.
Therefore, when α1-antitrypsin, α1-antichymotrypsin, and ulinastatin were allowed to act on physiological saline to which 500 leukocytes / μl had been added, and the reaction was examined by the test strip method, FIG. 10 (b) ( c)
The result of (d) was obtained. From this result, the inhibitor is α
It is concluded that there is a high possibility that the urinary trypsin inhibitor is not 1-antitrypsin or α1-antichymotrypsin, but urinastantin (molecular weight of about 60,000) that is also present in the urine of healthy subjects.

Since it is expected that the reaction of the test strip method will be different between the leukocytes in the state of cells and the leukocytes in the state of disintegration, ultrasonic waves were detected for the inconsistent urine in FIG. 2 containing a high concentration of the inhibitor. The reaction characteristics of the test strip method were examined for the case where 500 leukocytes / μl treated were added to the supernatant of the inconsistent urine and the case where 500 leukocytes / μl untreated were added. The results are as shown in FIG. 11, and in the inconsistent urine, the esterase derived from the disrupted leukocytes does not show activity (left column in FIG. 11), but the esterase activity in the leukocytes is completely disintegrated on the test paper. It was revealed that there was little inhibition (right column of FIG. 11). It is considered that this is the reason why the results obtained by the test strip method are relatively consistent with the white blood cell count in the urine sediment.

[Effectiveness of the Present Invention] As described above, it has been clarified that the sedimentoscopy method has a problem of leukolysis and the test strip method has a problem of urinary trypsin inhibitor. Therefore, lactoferrin Lf and myeloperoxidase M
Next, the effectiveness of urinary leukocyte detection by PO was examined. The correlation diagram of FIG. 12 (a) shows that myeloperoxidase MPO was obtained for randomly selected urine samples (daily samples).
It shows the correlation between the concentration and the lactoferrin Lf concentration. According to the result of FIG. 12A, a group in which the MPO concentration and the Lf concentration have a correlation and a group in which only the Lf concentration has a high value are recognized. In the group showing a high Lf concentration alone, no example was observed in which urine contained 10 / HPF or more leukocytes.

Next, 1 to 1000 leukocytes / μl are added to the urine sample showing low MPO concentration and Lf concentration.
When added, a good correlation was observed between both proteins (Fig. 12 (b)). From the above results, in FIG. 12A, it is considered that the group in which the MPO concentration and the Lf concentration have a correlation reflects the white blood cell count, while the group in which only the Lf concentration has a high value has no leukocytes. It is considered that there is some other cause that only the Lf concentration becomes extremely high. Figure 1
3 shows the stability of MPO in urine and the addition recovery rate. As shown in FIG. 13 (a), MPO in urine was 3
The residual rate is 95% or more even after standing at 7 ° C. for 24 hours. Further, as shown in FIG. 13B, the average recovery rate is 9
The rate is 6.3%, and it is considered that there is almost no possibility of false negatives due to cell disruption and enzyme activity inhibition observed in the sediment microscopy and test strip methods.

FIG. 14 (a) shows 640 cases of elementary school 4 years or younger (400 cases of school age or younger, 120 cases of elementary school 1.2 years, 1 year of elementary school 3.4 years), which are considered to have few abnormalities in the urinary tract.
Urine MPO values were obtained for 20 cases). The result was 80 ± 200 ng / ml (mean ± 2SD), and the cutoff value was 250 ng / ml. In addition, M
The PO value is measured by the following ELISA method. (1) The final concentration of 0.05 was added to the wells of the Mypro plate on which the polyclonal antibody against anti-human myeloperoxidase was immobilized.
50 μl of urine sample containing 10% surfactant (TritonX100)
To sample. (2) After incubating at 37 ° C. for 1 hour, the plate is washed with pure water, and 100 μl of an alkaline phosphatase (ALP) -labeled antibody diluted to a concentration of 2.5 μl / ml is poured into the well of the microplate. (3) 3
After incubating at 7 ° C. for 1 hour, the mixture is washed with pure water and 100 μl of KIND-KING method ALP substrate is dispensed. (4) After incubating at 37 ° C for 30 minutes, 100 μl of ALP color development solution was added to 510/630.
The concentration of myeloperoxidase MPO is calculated by photometry at nm.

Next, when the relationship between the number of neutrophils in urine and the MPO concentration was determined, the results shown in FIG. 15 were obtained. That is, an appropriate amount of white blood cells was added to physiological saline containing a final concentration of 0.05% of a surfactant (Triton X100) up to 300 cells / μl, and the mixture was double-diluted with a physiological saline containing a surfactant, followed by ELISA. The myeloperoxidase concentration was measured by the method to determine the relationship with the white blood cell count. According to the result of FIG. 15, the number of white blood cells corresponding to the MPO cutoff value of 250 ng / ml is about 1.
The number was 00 / μl. Cutoff value of test paper method is 10
~ 30 / μl, equivalent to 5-15 of sediment microscopy
This is about a three-fold increase compared to individual / HPF. From this result,
It is suggested that a large number of leukocytes have already been disrupted in urine, which also proves that the present invention is preferable as a method for detecting leukocytes in urine, which is capable of measuring disrupted leukocytes.

[Study that only Lf concentration in urine is high] Next, in FIG. 12 (a), a group was examined in which only Lf concentration was extremely high even though there were no leukocytes. In acute glomerulonephritis, horse cedar nephritis, or acute pyelonephritis after streptococcal infection mainly composed of inflammatory cells, neutrophils are activated by immune complexes and the like, releasing active oxygen to the outside of the cell and damaging the kidneys. It is said. Also, MPO of neutrophils consists of H ₂ O ₂ and halogens (chlorine, iodine, etc.)
Produces strong active oxygen such as HOCl or HOI that easily reacts with bacterial membranes. Since this MPO is a basic protein, it attaches to the acidic hyaluronic acid (which works to prevent the protein from being released from the blood into the urine), which is abundant in the glomerular basement membrane, and H ₂ O ₂ is present there. Then, it has been reported that proteinuria appears.

The onset age of nephritis found by school mass urinalysis is said to be 11 to 15 years in 80% of cases, but similar results were obtained in the study by the present inventor. In the 4th grade (10 years old and younger), there was no case in which only lactoferrin showed a high value (FIG. 16). Note that FIG.
Fig. 7 is a summary of the 1360 cases shown in Fig. 16 into two groups of 5th grade and above and 4th grade and below.
Of 300 ng / ml or more and MPO / LF of 0.
40 cases (40/1360 = 2.9)
%)Is recognized. Since it is said that transferrin (Tf) appears at the earliest stage with nephritis, next, (a) elementary school 4 years or older, Lf, MPO 50
428 cases of ng / ml or less, (b) 320 cases of 5 years or more in elementary school and Lf and MPO of 50 ng / ml or less,
(C) Elementary school 5 years or older and only Lf was high 40
Example (MPO / LF is 0..Lf when Lf is 300 ng / ml or more.
Tf concentration and Lf concentration were examined for 3 groups (less than 2).

FIG. 18 shows the results, and it was clarified that the Lf concentration and the Tf concentration are uncorrelated in any of the groups, and that the Tf concentration is not high even in the group of FIG. 18 (c). . Note that FIG. 19 is similar to FIG. 18 (a) (b) (c).
3 shows the average value of Tf concentration and t-test result, etc., and there was no significant difference between the urinary trunk ferrin concentrations in the 3 groups, Lf ≧ 300 ng / ml, MP
It is considered that proteinuria did not appear even in the group of O / LF <0.2. Considering the above points, Lf ≧ 30
The group of FIG. 18 (c) in which 0 ng / ml and MPO / LF <0.2, that is, the group in which only the Lf concentration is high in FIG. 12 (a), is in the initial stage of nephritis before the protein leaks into the urine. Is considered to reflect. Therefore, when the MPO concentration in urine and the Lf concentration in urine are measured and only the Lf concentration in urine shows a high value, the possibility of nephritis in the initial stage is estimated.

[0020]

As described above, in the present invention, leukocytes in urine are detected by measuring the myeloperoxidase concentration in urine. The cell count method is prone to false negatives because urinary leukocyte breakdown is severe, and the enzyme activity measurement method is inhibited by urinary trypsin inhibitors, but the myeloperoxidase protein level is stable in urine. It is excellent, has little effect of inhibitors, and exerts an excellent effect of being able to detect even disrupted leukocytes.

[Brief description of drawings]

FIG. 1 is a diagram comparing the concentration of neutrophil-derived protein in urine with the white blood cell count of urinary sediment.

FIG. 2 is a diagram comparing the concentration of neutrophil-derived protein in urine with the measurement result of the test strip method.

FIG. 3 is a diagram showing the distribution of leukocyte lysis rate in urine for 75 daily samples.

FIG. 4 is a drawing showing the relationship between the properties of urine samples and leukolysis rate.

FIG. 5: Time, temperature, and
And the relationship with pH.

FIG. 6 is a drawing showing the mechanism of urinary leukocyte breakdown.

FIG. 7 is a drawing showing the degree of inhibition of leukolysis by an inhibitor.

FIG. 8 is a drawing showing the degree of inhibition of esterase activity.

FIG. 9 is a drawing showing the molecular weights of inhibitors.

FIG. 10 is a drawing showing the results of examination of inhibitors for the test strip method.

FIG. 11 is a drawing showing the reaction characteristics of the test strip method for leukocytes in urine containing an inhibitor.

FIG. 12 is a drawing showing the relationship between myeloperoxidase concentration and lactoferrin concentration.

FIG. 13 is a drawing showing the stability of MPO in urine and the result of an addition recovery test.

FIG. 14 is a drawing showing the distribution of MPO values in urine of healthy subjects.

FIG. 15 is a drawing showing the relationship between the number of neutrophils and MPO concentration.

FIG. 16 is a drawing showing the relationship between MPO concentration and Lf concentration for 1360 cases.

FIG. 17 shows the results of FIG. 16 for groups of 5th grade and above and 4th grade.
It is a drawing that is summarized in groups of grades below.

FIG. 18: Lf, MPO ≦ 50 ng / m in 4th grade and below
l, Lf, MPO ≤ 50 ng / ml in elementary school 5 years or older, Lf ≥ 300 ng / ml, MPO / LF <in elementary school 5 years or older
It is drawing which shows the relationship of Tf concentration and Lf concentration about three groups of 0.2.

FIG. 19 shows three groups of FIGS. 18 (a) (b) (c),
It is drawing which showed the average value of Tf density | concentration, the t test result, etc.

Continuation of front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 33/48-33/98

Claims (3)

(57) [Claims] 1. A urinary tract infection or urinary tract inflammation characterized by detecting urinary leukocytes including depleted leukocytes by measuring urinary myeloperoxidase concentration.
Method for sexually transmitted diseases . 2. An EL using an anti-human polyclonal antibody
The urinary tract infection according to claim 1, wherein the urinary myeloperoxidase concentration is measured by the ISA method.
Or urinary tract inflammatory disease test method . 3. The urinary myeloperoxidase concentration is 25.
The urinary tract infection according to claim 1 or 2, wherein an abnormal value of about 0 ng / ml or more is determined.
Method for testing urinary tract inflammatory disease .