JP2004150832A - Standard particle suspension for flow cytometer - Google Patents
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【０００１】
【発明の属する技術分野】
本発明は、フローサイトメトリを利用した細菌検査装置の精度管理あるいはキャリブレーションに利用する粒子懸濁液に関する。
【０００２】
【従来の技術】
尿中の細菌をフローサイトメトリにより検出する方法はすでに知られている。尿試料中の細菌を蛍光色素により染色し、フローサイトメータのフローセルに流して、フローセル中を流れる細菌に励起光を照射し、細菌より発せられる散乱光および／または蛍光を検出する。検出された散乱光信号および／または蛍光信号は、解析用アルゴリズムによって、測定試料中に共存する他の成分から弁別され計数される。
【０００３】
ところで、解析用アルゴリズムを用いて、細菌を正確に弁別計数するためには、フローサイトメータを測定に適した状態に恒常的に維持しておく必要がある。具体的には、光学系においては、散乱光や蛍光を受光する受光部の感度、光軸など、さらに測定用試料の調製も自動化されているものでは、試料や試薬の定量、反応温度や反応時間など、染色システムが適正に維持されていなければならない。したがって、測定に先立って、フローサイトメータが測定に適正な状態であるかをチェックし（精度管理）、必要であれば、装置を適切な状態にキャリブレーションしなければならない。
【０００４】
フローサイトメータのキャリブレーションを行うために、微小球または微小ビーズなどの人工粒子を用いる方法が知られている（例えば特許文献１、特許文献２、特許文献３参照）。
【０００５】
【特許文献１】
米国特許第４，７０４，８９１号明細書
【特許文献２】
米国特許第５，０７３，４９７号明細書
【特許文献３】
特開平９−１９６９１６号公報
【０００６】
【発明が解決しようとする課題】
フローサイトメータの染色システムを管理するためには、精度管理やキャリブレーションに用いる粒子は、測定対象とする細菌と同程度の散乱光強度を有し、測定に使用する染色液で染色したときに細菌と同程度の蛍光強度を有することが好ましい。またキャリブレーションを行うには、フローサイトメーターから得られる各信号の分布が均一になっている事がより望ましい。
【０００７】
ところで、尿中の細菌を染色する際には、特開２００１−２５８５９０号公報のように共存する夾雑物の影響を回避するために低いｐＨで染色する方が好ましい。しかし、低いｐＨにおいて細菌と同程度に染色され、かつ、細菌と同程度の散乱光強度を有する人工粒子は見出せていない。
【０００８】
本発明は、染色条件が低いｐＨにおいても精度管理やキャリブレーションが可能な標準粒子懸濁液を提供することを目的とする。
【０００９】
【課題を解決するための手段】
本発明のフローサイトメータ用標準粒子懸濁液は、ポリスチレン系重合体粒子とポリ酢酸ビニル粒子を含むことを特徴とする。
【００１０】
【発明の実施の形態】
本発明のポリスチレン系重合体粒子は、細菌と同程度の散乱光強度を有するもので、ポリスチレン粒子、ポリスチレン共重合体粒子等が使用できる。平均粒径は、０．５〜２．０μｍのものが好適に選択される。粒子の大きさは装置が設定する感度に合わせて、適宜選択され得る。また、装置のキャリブレーションを行うためには、粒子の粒径が均一で分布が単峰であり、粒子濃度は１０個／μｌ以上あることが好ましい。
【００１１】
また本発明のポリ酢酸ビニル粒子は、染色後、細菌に近い蛍光強度を示すもので、平均粒径は５〜８μｍのものが好適に選択される。粒子の大きさは装置が設定する感度に合わせて、適宜選択され得る。また、上記のポリスチレン系重合体粒子同様、粒径分布は均一であり、粒子濃度は１０個／μｌ以上あることが好ましい。なお、細菌の染色に用いられる蛍光色素は、分子中に陽電荷を有し、ｐＨ２．５〜４．５で使用でき、好適には、特開２００１−２５８５９０号公報に記載されているものが使用できる。この陽電荷とポリ酢酸ビニル粒子表面の負電荷官能基とが反応して、蛍光を帯びるようになる。
【００１２】
本発明のフローサイトメータ用標準粒子懸濁液には、計数値を正確に出すための工夫として、粒子凝集を防止する事が望ましい。このため懸濁液には粒子凝集防止剤を添加することが好ましい。粒子凝集防止剤としては、ポリビニルアルコール、界面活性剤が挙げられ、ポリオキシエチレン系ノニオン性界面活性剤が好ましい。使用濃度範囲は、０．０５〜０．５％（ｗ／ｖ）が好適である。
【００１３】
なお、赤色レーザーを光源とするフローサイトメータで、尿中の細菌を測定する場合には、尿試料の屈折率が高いので、フローセルに流すシース液の屈折率も尿試料に合わせて高くすることが好ましい。こうすることによって、散乱光信号の揺らぎが抑えられ、正確な測定が可能になる。したがって、このような装置では、精度管理やキャリブレーションに用いる粒子懸濁液もシース液に合わせて屈折率を調整することが好ましい。
【００１４】
屈折率を調整するには、水溶液中で溶解し且つ非電解質の有機物が好ましい。例えば、糖類、尿素、グリコール類など挙げられるが、中でもグリコール類が粒子懸濁液の屈折率調節剤として好適である。特にエチレングリコールが好ましい。使用濃度範囲は、測定するサンプルの希釈倍率や測定温度によって、適宜最適な濃度に調整しても良いが、好ましくは、６〜１２％（ｗ／ｖ）が良い。エチレングリコールは粒子凝集防止剤を長期間安定させる増粘剤として効果がある点で優れている。
【００１５】
本発明のフローサイトメータ用粒子懸濁液を用いて、フローサイトメータの精度管理あるいはキャリブレーションを行うには、複数の計数値及び感度パラメータの値を確認する必要がある。装置が安定した状態である事を証明するには、これら粒子懸濁液の計数値及び感度パラメーターを継続して管理し、ある一定の範囲にある事を確認する必要がある。
【００１６】
ところで、細菌を測定するフローサイトメータは、臨床サンプル中の細菌数だけでなく、白血球数を計数することで、感染の状態をより詳細に把握できる。細菌の大きさは、菌種やサンプルの状態によっても様々に変化するが、一般的に０．５μｍから２，３μｍと小さく、白血球は７〜８μｍと大きい。したがって、細菌と白血球を同時に計数する場合において、細菌を検出するには、前方散乱光の感度を白血球測定時よりも１５〜２０倍程度高くするのが好ましい。
【００１７】
細菌を捉える二次元分布図（スキャッタグラム）で使用するパラメータは、図１及び図２に示すように▲１▼高感度の前方散乱光強度、高感度の前方散乱光強度幅及び、▲３▼蛍光強度が好ましい。白血球を捉えるスキャッタグラムで使用するパラメーターは、図３及び図４に示すように▲１▼低感度の前方散乱光強度、▲２▼低感度の前方散乱光強度幅及び▲３▼蛍光強度が好ましい。このように複数のパラメータが存在するため、これらの感度に変化がないか、又は変化があった場合に、装置の状態を一定にするために調整する必要が生じる。
【００１８】
【実施例】
（粒子懸濁液）
平均粒径１μｍポリスチレン粒子 ５００個／μｌ
平均粒径約６μｍポリ酢酸ビニル粒子 ５００個／μｌ
グリシン ５０ｍＭ
ＨＣｌ １．２ｍＭ
エチレングリコール １０．６ｗ／ｖ％
レオドールＴＷ−Ｏ１２０ ０．１ｗ／ｖ％
（ポリエチレングリコール（２０）ソルビタンエステル）
溶媒（精製水）
（希釈液）
クエン酸 ９２．３ｍＭ
ＮａＯＨ ０．７５ｇ／ｌ
テトラデシルトリメチルアンモニウムブロマイド ０．１ｗ／ｖ％
Ｎａ２ＳＯ４ ９０ｍＭ
スルファミン酸ナトリウム １００ｍＭ
溶媒（精製水）
（染色液）
以下の蛍光色素Ａ ４０ｐｐｍ（エチレングリコール溶液）
【化１】
上記粒子懸濁液、大腸菌含有試料、白血球含有試料各５０μｌに、上記の希釈液３４０μｌ及び染色液を色素Ａの最終濃度が１ｐｐｍになるようにそれぞれ添加し、４０℃、２０秒間反応させ、赤色半導体レーザーを光源とするフローサイトメータで散乱光及び蛍光の測定を行った（分析容量６．０μｌ）。細菌を検出するために、前方散乱光感度を白血球測定時より１７倍高くして測定を行った。
【００２０】
測定の結果得られた各パラメータのスキャッタグラム及び粒度分布を図１から図６に示す。
【００２１】
大腸菌は、高感度前方散乱光強度が約１５〜７０ｃｈの範囲、蛍光強度が約５０〜２００ｃｈの範囲、高感度前方散乱光強度幅は、約２５〜８０ｃｈの範囲で分布していた（図１及び図２参照）。これらの範囲が含まれるように蛍光強度−高感度前方散乱光強度スキャッタグラム及び高感度前方散乱光強度幅−高感度前方散乱光強度スキャッタグラム上でウインドウをそれぞれ作成し、次に両方のウインドウ内に入るという条件を満たす粒子数を計数し、細菌数とする。
【００２２】
また、白血球は、低感度前方散乱光強度が約５０〜１２０ｃｈの範囲、蛍光強度がほぼ２５５ｃｈで飽和状態、低感度前方散乱光強度幅は、約５０〜８０ｃｈの範囲で分布していた（図３及び図４参照）。これらの範囲が含まれるように蛍光強度−低感度前方散乱光強度スキャッタグラム及び低感度前方散乱光強度幅−低感度前方散乱光強度スキャッタグラム上でウインドウを作成し、次に両方のウインドウ内に入るという条件を満たす粒子数を計数し、白血球数とする。
【００２３】
一方、このとき、１μｍポリスチレン粒子は、高感度前方散乱光強度が約８５〜９５ｃｈの範囲で分布し、ピーク値は８９ｃｈであり、また、高感度前方散乱光強度幅は約６０〜６４ｃｈの範囲で分布し、ピーク値は６２ｃｈであった（図５参照）。また、粒度分布は単峰を示した。
【００２４】
また、約６μｍポリ酢酸ビニル粒子は、低感度前方散乱光強度が約１９５〜２１０ｃｈの範囲で分布し、ピーク値は２０２ｃｈであり、低感度前方散乱光強度幅は約８５〜９５ｃｈの範囲で分布し、ピーク値は９１ｃｈであった。蛍光強度は４５〜７５ｃｈの範囲で分布し、ピーク値は６０ｃｈであった（図６参照）。粒度分布は単峰を示した。
【００２５】
精度管理やキャリブレーションは、あらかじめ上記の各ピーク値に対して、ある一定の許容幅を設定しておき、その設定値範囲内であるかどうかを確認することによって実施することができる。
【発明の効果】
【００２６】
本発明によれば、１回の測定で、各計数値と測定パラメーター値を一度に得る事ができ、装置の状態をより簡便に把握できる。また、たとえ装置の状態が悪いことが判明しても、本発明の粒子懸濁液を測定することによって、予め期待される値に感度を合わせることが容易である。
【図面の簡単な説明】
【図１】細菌を含む試料を測定したときの蛍光強度−高感度前方散乱光強度を示すスキャッタグラムである。
【図２】細菌を含む試料を測定したときの高感度前方散乱光強度幅−高感度前方散乱光強度を示すスキャッタグラムである。
【図３】白血球を含む試料を測定したときの蛍光強度−低感度前方散乱光強度を示すスキャッタグラムである。
【図４】白血球を含む試料を測定したときの低感度前方散乱光強度幅−低感度前方散乱光強度を示すスキャッタグラムである。
【図５】本発明の粒子懸濁液を測定したときの高感度前方散乱光強度幅−高感度前方散乱光強度を示すスキャッタグラム、高感度前方散乱光強度ヒストグラム及び高感度前方散乱光強度幅ヒストグラムである。
【図６】本発明の粒子懸濁液を測定したときの蛍光強度−低感度前方散乱光強度スキャッタグラム、低感度前方散乱光強度幅−低感度前方散乱光強度スキャッタグラム、蛍光強度ヒストグラム、低感度前方散乱光強度ヒストグラム及び低感度前方散乱光強度幅ヒストグラムである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a particle suspension used for quality control or calibration of a bacteria test device using flow cytometry.
[0002]
[Prior art]
Methods for detecting bacteria in urine by flow cytometry are already known. Bacteria in the urine sample are stained with a fluorescent dye, flow into a flow cell of a flow cytometer, and the bacteria flowing in the flow cell are irradiated with excitation light to detect scattered light and / or fluorescence emitted from the bacteria. The detected scattered light signal and / or fluorescent signal are discriminated from other components coexisting in the measurement sample and counted by the analysis algorithm.
[0003]
Incidentally, in order to accurately discriminate and count bacteria using an analysis algorithm, it is necessary to constantly maintain a flow cytometer in a state suitable for measurement. Specifically, in an optical system, the sensitivity of the light receiving unit that receives scattered light and fluorescence, the optical axis, etc. The staining system, such as time, must be properly maintained. Therefore, before the measurement, it is necessary to check whether the flow cytometer is in a proper state for the measurement (quality control) and, if necessary, to calibrate the apparatus to a proper state.
[0004]
There is known a method of using artificial particles such as microspheres or microbeads to calibrate a flow cytometer (for example, see Patent Literature 1, Patent Literature 2, Patent Literature 3).
[0005]
[Patent Document 1]
US Patent No. 4,704,891 [Patent Document 2]
US Pat. No. 5,073,497 [Patent Document 3]
JP-A-9-196916
[Problems to be solved by the invention]
In order to manage the staining system of the flow cytometer, the particles used for quality control and calibration have the same scattered light intensity as the bacteria to be measured, and are stained with the staining solution used for measurement. It is preferable to have the same fluorescence intensity as that of bacteria. To perform calibration, it is more desirable that the distribution of each signal obtained from the flow cytometer is uniform.
[0007]
Meanwhile, when staining bacteria in urine, it is preferable to stain at a low pH in order to avoid the influence of coexisting contaminants as in JP-A-2001-258590. However, no artificial particles have been found which are stained to the same extent as bacteria at low pH and have the same scattered light intensity as bacteria.
[0008]
An object of the present invention is to provide a standard particle suspension capable of performing quality control and calibration even at a low pH under staining conditions.
[0009]
[Means for Solving the Problems]
The standard particle suspension for a flow cytometer of the present invention is characterized by containing polystyrene-based polymer particles and polyvinyl acetate particles.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
The polystyrene-based polymer particles of the present invention have the same scattered light intensity as bacteria, and polystyrene particles, polystyrene copolymer particles and the like can be used. Those having an average particle size of 0.5 to 2.0 μm are suitably selected. The size of the particles can be appropriately selected according to the sensitivity set by the device. In order to calibrate the apparatus, it is preferable that the particle diameter is uniform and the distribution is unimodal, and the particle concentration is 10 particles / μl or more.
[0011]
The polyvinyl acetate particles of the present invention exhibit a fluorescence intensity close to that of bacteria after staining, and those having an average particle size of 5 to 8 μm are suitably selected. The size of the particles can be appropriately selected according to the sensitivity set by the device. Further, like the above-mentioned polystyrene-based polymer particles, the particle size distribution is preferably uniform, and the particle concentration is preferably 10 particles / μl or more. The fluorescent dye used for staining bacteria has a positive charge in the molecule and can be used at pH 2.5 to 4.5. Preferably, those described in JP-A-2001-258590 are used. Can be used. The positive charge reacts with the negatively-charged functional group on the surface of the polyvinyl acetate particles, and becomes fluorescent.
[0012]
In the standard particle suspension for a flow cytometer of the present invention, it is desirable to prevent particle aggregation as a device for accurately obtaining a count value. Therefore, it is preferable to add a particle aggregation inhibitor to the suspension. Examples of the particle aggregation preventing agent include polyvinyl alcohol and a surfactant, and a polyoxyethylene-based nonionic surfactant is preferable. The concentration range for use is preferably 0.05 to 0.5% (w / v).
[0013]
When measuring bacteria in urine with a flow cytometer using a red laser as the light source, the refractive index of the urine sample is high, so the refractive index of the sheath liquid flowing through the flow cell should also be increased according to the urine sample. Is preferred. By doing so, the fluctuation of the scattered light signal is suppressed, and accurate measurement becomes possible. Therefore, in such an apparatus, it is preferable to adjust the refractive index of the particle suspension used for accuracy control and calibration in accordance with the sheath liquid.
[0014]
In order to adjust the refractive index, an organic substance which is soluble in an aqueous solution and is a non-electrolyte is preferable. For example, saccharides, urea, glycols and the like can be mentioned. Among them, glycols are preferable as a refractive index regulator of the particle suspension. Particularly, ethylene glycol is preferred. The working concentration range may be appropriately adjusted to an optimum concentration depending on the dilution ratio of the sample to be measured and the measurement temperature, but is preferably 6 to 12% (w / v). Ethylene glycol is excellent in that it is effective as a thickener for stabilizing the particle aggregation inhibitor for a long period of time.
[0015]
In order to control the accuracy or calibrate the flow cytometer using the particle suspension for a flow cytometer of the present invention, it is necessary to confirm a plurality of count values and sensitivity parameter values. In order to prove that the apparatus is in a stable state, it is necessary to continuously control the count values and sensitivity parameters of these particle suspensions and confirm that the values are within a certain range.
[0016]
By the way, a flow cytometer for measuring bacteria can grasp the state of infection in more detail by counting the number of white blood cells as well as the number of bacteria in a clinical sample. The size of the bacterium varies variously depending on the type of the bacterium and the state of the sample, but is generally as small as 0.5 μm to 2.3 μm, and the size of the leukocyte is as large as 7 to 8 μm. Therefore, when bacteria and leukocytes are counted simultaneously, in order to detect bacteria, it is preferable that the sensitivity of the forward scattered light be about 15 to 20 times higher than that at the time of leukocyte measurement.
[0017]
As shown in FIGS. 1 and 2, the parameters used in the two-dimensional distribution chart (scattergram) capturing bacteria are: (1) high-sensitivity forward scattered light intensity, high-sensitivity forward scattered light intensity width, and (3). Fluorescence intensity is preferred. As shown in FIGS. 3 and 4, the parameters used in the scattergram capturing leukocytes are preferably (1) low-sensitivity forward scattered light intensity, (2) low-sensitivity forward scattered light intensity width, and (3) fluorescence intensity. . Since there are a plurality of parameters as described above, there is no change in these sensitivities, or if there is a change, it is necessary to adjust the state of the apparatus to be constant.
[0018]
【Example】
(Particle suspension)
Average particle size 1μm polystyrene particles 500 particles / μl
Average particle size about 6μm polyvinyl acetate particles 500 / μl
Glycine 50 mM
HCl 1.2 mM
Ethylene glycol 10.6w / v%
REODOL TW-O120 0.1w / v%
(Polyethylene glycol (20) sorbitan ester)
Solvent (purified water)
(Diluted solution)
92.3 mM citric acid
NaOH 0.75 g / l
Tetradecyltrimethylammonium bromide 0.1w / v%
Na 2 SO 4 90 mM
Sodium sulfamate 100mM
Solvent (purified water)
(Staining solution)
The following fluorescent dye A 40ppm (ethylene glycol solution)
Embedded image
To each of the particle suspension, E. coli-containing sample, and leukocyte-containing sample, 50 μl, 340 μl of the diluent and the staining solution were added so that the final concentration of the dye A was 1 ppm, and the mixture was allowed to react at 40 ° C. for 20 seconds. The scattered light and the fluorescence were measured by a flow cytometer using a semiconductor laser as a light source (analysis volume: 6.0 μl). In order to detect bacteria, the measurement was performed with the forward scattered light sensitivity 17 times higher than that in the white blood cell measurement.
[0020]
The scattergram and particle size distribution of each parameter obtained as a result of the measurement are shown in FIGS.
[0021]
Escherichia coli had a high sensitivity forward scattered light intensity of about 15 to 70 ch, a fluorescence intensity of about 50 to 200 ch, and a high sensitivity forward scattered light intensity range of about 25 to 80 ch (FIG. 1). And FIG. 2). A window is created on each of the fluorescence intensity-high-sensitivity forward scattered light intensity scattergram and the high-sensitivity forward scattered light intensity scattergram-high-sensitivity forward scattered light intensity scattergram so that these ranges are included. Count the number of particles that satisfy the condition of entry into the bacteria count.
[0022]
In addition, the leukocytes had a low-sensitivity forward scattered light intensity in a range of about 50 to 120 ch, a fluorescence intensity of about 255 ch and were saturated, and a low-sensitivity forward scattered light intensity range was in a range of about 50 to 80 ch (FIG. 3 and FIG. 4). A window is created on the fluorescence intensity-low sensitivity forward scattered light intensity scattergram and low sensitivity forward scattered light intensity scattergram-low sensitivity forward scattered light intensity scattergram so that these ranges are included, and then a window is created in both windows. The number of particles that satisfy the condition for entry is counted and defined as the number of white blood cells.
[0023]
On the other hand, at this time, the 1 μm polystyrene particles have a highly sensitive forward scattered light intensity distributed in a range of about 85 to 95 ch, a peak value of 89 ch, and a high sensitive forward scattered light intensity range of about 60 to 64 ch. And the peak value was 62 ch (see FIG. 5). The particle size distribution showed a single peak.
[0024]
In addition, about 6 μm polyvinyl acetate particles have a low sensitivity forward scattered light intensity distributed in a range of about 195 to 210 ch, a peak value of 202 ch, and a low sensitivity forward scattered light intensity range distributed in a range of about 85 to 95 ch. The peak value was 91 ch. The fluorescence intensity was distributed in the range of 45 to 75 ch, and the peak value was 60 ch (see FIG. 6). The particle size distribution showed a single peak.
[0025]
Accuracy management and calibration can be performed by setting a certain allowable width in advance for each of the above-mentioned peak values, and confirming whether the value falls within the set value range.
【The invention's effect】
[0026]
According to the present invention, each count value and measurement parameter value can be obtained at once by one measurement, and the state of the device can be grasped more easily. Even if it is found that the state of the apparatus is bad, it is easy to adjust the sensitivity to an expected value by measuring the particle suspension of the present invention.
[Brief description of the drawings]
FIG. 1 is a scattergram showing fluorescence intensity-high-sensitivity forward scattered light intensity when a sample containing bacteria is measured.
FIG. 2 is a scattergram showing high sensitivity forward scattered light intensity width-high sensitivity forward scattered light intensity when a sample containing bacteria is measured.
FIG. 3 is a scattergram showing fluorescence intensity-low-sensitivity forward scattered light intensity when a sample containing leukocytes is measured.
FIG. 4 is a scattergram showing low sensitivity forward scattered light intensity width-low sensitivity forward scattered light intensity when a sample containing leukocytes is measured.
FIG. 5 is a scattergram showing high-sensitivity forward scattered light intensity—high-sensitivity forward scattered light intensity histogram and high-sensitivity forward scattered light intensity width when measuring the particle suspension of the present invention. It is a histogram.
FIG. 6: Fluorescence intensity—low-sensitivity forward scattered light intensity scattergram, low-sensitivity forward scattered light intensity width—low-sensitivity forward scattered light intensity scattergram, fluorescence intensity histogram, low intensity It is a sensitivity forward scattered light intensity histogram and a low sensitivity forward scattered light intensity width histogram.
Claims (7)
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