JP4946047B2 - Particle standard reagent for formed component classifier - Google Patents
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Description
本願発明は、生体試料中の有形成分を分類し、個数計測する自動合焦機能が装備されることを特徴とする有形成分分類装置の精度管理あるいはキャリブレーションに使用する粒子標準試薬に関するものである。 The present invention relates to a particle standard reagent used for accuracy control or calibration of a formed component classification device, which is equipped with an automatic focusing function for classifying formed components in a biological sample and counting the number of formed components. It is.
従来から、生体試料中、特に尿中の有形成分の分析には、フローサイトメーターを用いて検体の塗布標本を作製せず生体試料に染色液を混和した後、懸濁させたままフローセルに流し、物理統計的方法や光学的方法などによって自動分析する装置があるのは公知であり、また、この装置が測定に適切な状態であるか精度管理したり、キャリブレーションを行なうことを目的とする粒子懸濁液は公知である（例えば、特許文献１）。
しかし、上記のフローサイトメーター法により自動分析する装置では、有形成分は常に流れているためそれぞれ焦点を合わせることは極めて困難であり、焦点は一定の位置に固定されている。また、フローセルはある程度厚みがある。これらのことより、上記のフローサイトメーター法では焦点が合わされた位置に流れてきた有形成分しか正確に測定できず、更には該位置から少し離れたところに有形成分が位置した場合は焦点がずれた分だけぼやけてしまい、合焦時とは異なった画像計測結果が得られ、誤った判断結果が得られることがある。 However, in the apparatus that automatically analyzes by the above flow cytometer method, the formed components are always flowing, so it is very difficult to focus on each, and the focus is fixed at a fixed position. In addition, the flow cell has a certain thickness. Based on these facts, the above flow cytometer method can accurately measure only the formed component that has flowed to the focused position, and if the formed component is located slightly away from the focused position, The amount of deviation is blurred and an image measurement result different from that at the time of in-focus is obtained, and an erroneous determination result may be obtained.
フローサイトメーター法の欠点を解決するために、生体試料を撮像する工程を有し、生体試料中の有形成分を撮像する際、焦点を自動で合わせる機能を装備することで焦点の合った鮮明な画像が得られることを特徴とする分析装置（例えば、特許文献２）が知られているが、鮮明な画像として得られるが故に、この分類装置を精度管理し、キャリブレーションを行なうための粒子はフローサイトメーター法に使用する粒子と比較して、形態学的に有形成分とより相同性があるものでなければならなく、これまで存在しなかった。 In order to solve the drawbacks of the flow cytometer method, it has a process of imaging a biological sample, and when imaging a formed component in a biological sample, it is equipped with a function that automatically adjusts the focus so that the focus is clear. Analyzing devices (for example, Patent Document 2) characterized in that a clear image can be obtained are known. However, since they are obtained as clear images, particles for controlling the accuracy of the classification device and performing calibration are known. Must be morphologically more homologous to the formed component compared to the particles used in the flow cytometer method and has not existed so far.
発明者は、鋭意検討した結果、粒子を非イオン性界面活性剤と共存させることで、粒子に染色液中の色素を均一に結合させるようにすることで、実際の血球成分と同様な撮像画像が得られ、それにより分類装置のプログラムを何ら改変することなく、精度管理し、キャリブレーションを行なうことが可能な本願発明を完成した。 As a result of intensive studies, the inventor made the particles coexist with a nonionic surfactant so that the dye in the staining solution is uniformly bound to the particles, thereby obtaining a captured image similar to an actual blood cell component. As a result, the present invention has been completed, in which the accuracy can be controlled and calibrated without modifying the classification device program.
すなわち本願発明の要旨は、以下の通りである。
［項１］透光板上の生体試料を撮像する手段を有し、かつ該手段において生体試料中の有形成分に焦点を自動的に合わせる自動合焦機能が装備される有形成分分類装置に使用する、粒子を含む標準試薬。
［項２］ 該粒子が、ポリスチレン、メラミン、ポリメタクリル酸、ガラスのうちから少なくとも１つ以上が選ばれる、項１記載の標準試薬。
［項３］さらに色素親和剤を含む、項２記載の標準試薬。
［項４］色素親和剤が、非イオン性界面活性剤から少なくとも１つ以上が選ばれる項３記載の標準試薬。
［項５］非イオン系界面活性剤が、ポリオキシエチレンソルビタン脂肪酸エステル系界面活性剤、ポリエチレングリコール系界面活性剤、ソルビタン脂肪酸エステル系界面活性剤、ポリオキシエチレンアルキルエーテル系界面活性剤から少なくとも１つ以上が選ばれる項４記載の標準試薬。
That is, the gist of the present invention is as follows.
[Item 1] A formed component classification apparatus having means for imaging a biological sample on a translucent plate and equipped with an automatic focusing function for automatically focusing on the formed component in the biological sample. Standard reagent containing particles for use in
[Item 2] The standard reagent according to Item 1, wherein the particles are at least one selected from polystyrene, melamine, polymethacrylic acid, and glass.
[Item 3] The standard reagent according to Item 2, further comprising a dye affinity agent.
[Item 4] The standard reagent according to item 3, wherein the dye affinity agent is at least one selected from nonionic surfactants.
[Item 5] The nonionic surfactant is at least one selected from a polyoxyethylene sorbitan fatty acid ester surfactant, a polyethylene glycol surfactant, a sorbitan fatty acid ester surfactant, and a polyoxyethylene alkyl ether surfactant. Item 5. The standard reagent according to Item 4, wherein one or more are selected.
本願発明は、透光板上の生体試料を撮像する手段を有し、かつ該手段において生体試料中の有形成分に焦点を自動的に合わせる自動合焦機能が装備されることを特徴とする有形成分分類装置において、粒子に染色液中の色素を均一に結合させるようにすることで、実際の血球成分と同様な撮像画像が得られ、それにより分類装置のプログラムを何ら改変することなく、精度管理し、キャリブレーションを行なう試薬を提供する。 The invention of the present application has means for imaging a biological sample on a translucent plate, and is equipped with an automatic focusing function for automatically focusing on a formed component in the biological sample in the means. In the formed component classification device, by uniformly binding the dye in the staining solution to the particles, a captured image similar to the actual blood cell component can be obtained, thereby making no modification to the classification device program Providing reagents for quality control and calibration.
本願発明は、生体試料中の有形成分を分類する装置において、分類装置のプログラムを何ら改変することなく、精度管理し、キャリブレーションを行なう試薬に関する。本願発明における生体試料とは、全血液、脊髄液、前立腺液、尿、腹水、関節液、涙液、唾液、血清、血漿など生体流体が含まれる。また、非生物学的性質の他の流体試料を分析することも可能である。 The present invention relates to a reagent for performing accuracy control and calibration without any modification of a program of a classification device in an apparatus for classifying a formed component in a biological sample. The biological sample in the present invention includes biological fluids such as whole blood, spinal fluid, prostate fluid, urine, ascites, joint fluid, tear fluid, saliva, serum, and plasma. It is also possible to analyze other fluid samples of non-biological nature.
本願発明における有形成分とは、赤血球、白血球などの血球、扁平上皮、移行上皮、尿細管上皮などの上皮細胞、硝子円柱、顆粒円柱、ろう様円柱などの円柱、細菌などが含まれる。 The formed component in the present invention includes blood cells such as erythrocytes and leukocytes, epithelial cells such as squamous epithelium, transitional epithelium and tubule epithelium, columns such as glass column, granule column and waxy column, bacteria and the like.
本願発明における粒子は、染色液中の色素との親和性が実在の有形成分とほぼ同程度になれば材質、形状、色、粒子径、濃度など何ら限定されない。材質は好ましくは、ポリスチレン、メラミン、ポリメタクリル酸、ガラスのうちから少なくとも１つ以上が選ばれ、より好ましくは単一のポリマーで形成されたものである。形状は好ましくは、球状粒子であり、より好ましくは、表面が円滑な球状粒子である。色は好ましくは、粒子全面が単一の色であり、より好ましくは白色である。粒子径は好ましくは、有形成分の平均的な大きさである直径５．０μｍ〜１５．０μｍであり、より好ましくは、直径６．０μｍ〜１２．０μｍの粒子径が均一な粒子である。濃度は、粒子同士が接触することがない濃度ならば限定されないが、好ましくは１００個／μｌ〜１０００個／μｌである。 The particle, the shape, the color, the particle diameter, and the concentration of the particles in the present invention are not limited as long as the affinity with the dye in the staining solution is approximately the same as the actual formed component. The material is preferably at least one selected from polystyrene, melamine, polymethacrylic acid, and glass, more preferably a single polymer. The shape is preferably spherical particles, and more preferably spherical particles having a smooth surface. The color is preferably a single color on the entire surface of the particle, more preferably white. The particle diameter is preferably 5.0 μm to 15.0 μm in diameter, which is an average size of the formed component, and more preferably particles having a uniform particle diameter of 6.0 μm to 12.0 μm. The concentration is not limited as long as the particles do not come into contact with each other, but is preferably 100 / μl to 1000 / μl.
本願発明において、染色液中の色素との親和性が実在の有形成分とほぼ同程度か否かの判断は、透光板中の生体試料を撮像する手段を有し、かつ該手段において生体試料中の有形成分に焦点を自動的に合わせる自動合焦機能が装備されることを特徴とする該有形成分分類装置が取得した撮影画像から判断される。具体的には、染色液中の色素により染色された粒子表面の色調、輝度、明度などが、該有形成分分類装置が実際の有形成分と認識する範囲にあれば染色液中の色素との親和性が実在の有形成分とほぼ同程度の粒子と判断する。この特徴を有し、かつ粒子径が目的の有形成分と同程度の粒子を形態学的に有形成分とより相同性がある粒子と定義する。 In the present invention, the determination as to whether or not the affinity with the dye in the staining solution is substantially the same as the actual formed component has a means for imaging the biological sample in the translucent plate, and the means Judgment is made from a photographed image acquired by the formation component classification apparatus, which is equipped with an automatic focusing function for automatically focusing on the formation component in the sample. Specifically, if the color tone, brightness, brightness, etc. of the surface of the particles dyed with the dye in the staining liquid are within the range that the component classification apparatus recognizes as an actual component, the dye in the staining liquid It is determined that the particles have an affinity of about the same level as that of the actual formed component. A particle having this characteristic and having a particle size similar to that of the target formed component is defined as a particle that is morphologically more homologous to the formed component.
本願発明における色素親和剤とは、粒子を造粒した際に粒子表面に付着した粒子の原料や有機溶媒などで形成された油膜層を親水化し、水中の色素が粒子表面に吸着できるようにする物質のことで、非イオン性界面活性剤が使用される。この非イオン性界面活性剤により、疎水性である粒子表面の界面張力を低下させ、染色液中の色素がより吸着できるようになり、実際の血球と同程度の均一かつ色彩の染色像を得ることが可能となる。濃度として好ましくは、０．０１ｗ／ｖ％〜０．５ｗ／ｖ％であり、この濃度範囲ならば、ミセル形成が起こらず、粒子同士の凝集および粒子と保存容器との吸着を防止する波及的効果が得られる。 The dye affinity agent in the present invention is to hydrophilize the oil film layer formed with the raw material of the particles or the organic solvent attached to the particle surface when the particles are granulated so that the dye in water can be adsorbed on the particle surface. By substance, a nonionic surfactant is used. This nonionic surfactant reduces the interfacial tension on the surface of the hydrophobic particles and allows the dye in the staining solution to be more adsorbed, resulting in a uniform and colored stained image similar to actual blood cells. It becomes possible. The concentration is preferably 0.01 w / v% to 0.5 w / v%. If the concentration is within this range, micelle formation does not occur, and spillover that prevents aggregation between particles and adsorption between the particles and the storage container is prevented. An effect is obtained.
［実施例］
（１）（粒子試薬の作製）
５０ｍＭリン酸緩衝液（ｐＨ７．０）に０．５ｗ／ｖ％になるようにＴｒｉｔｏｎ （登録商標） Ｘ−１００を添加し、１００個〜２００個／５０視野（３００個〜７０ ０個／μｌに相当）になるように平均粒子径１０μｍのポリメタクリル酸粒子を懸濁し た。
（２）（粒子試薬の測定）
尿中有形成分分析装置「Ｕ−ＳＣＡＮＮＥＲ（登録商標）」にて粒子を５０視野撮像し、同分析装置の解析プログラムによる自動計測を実施した。
[Example]
(1) (Preparation of particle reagent)
Triton (registered trademark) X-100 is added to 50 mM phosphate buffer (pH 7.0) so as to be 0.5 w / v%, and 100 to 200/50 fields (300 to 700 / μl). The polymethacrylic acid particles having an average particle diameter of 10 μm were suspended so that
(2) (Measurement of particle reagent)
Particles of 50 visual fields were imaged with a urine sediment analyzer “U-SCANNER (registered trademark)”, and automatic measurement was performed using an analysis program of the analyzer.
［比較例］
実施例の（２）の工程で、尿中有形成分分析装置「Ｕ−ＳＣＡＮＮＥＲ（登録商標）」にて粒子を５０視野撮像し、その画像を目視で計測した以外は実施例と同様に測定を実施した。
[Comparative example]
In the step (2) of the example, measurement was performed in the same manner as in the example except that 50 visual fields of the particles were imaged with the urine particle analyzer “U-SCANNER (registered trademark)” and the image was visually measured. Carried out.
５重測定を実施した結果を表１に示す。 Table 1 shows the results of the 5-fold measurement.
認識率（目視での計測数に対する自動計測数の割合）は、９５〜１００％と高く、ほぼ全てを見落とすことなく認識できた。 The recognition rate (ratio of the number of automatic measurements to the number of measurements by visual observation) was as high as 95 to 100%, and almost all could be recognized without overlooking.
本願発明は、透光板上もしくはフローセル中の生体試料を撮像する手段を有し、かつ該手段において生体試料中の有形成分に焦点を自動的に合わせる自動合焦機能が装備されることを特徴とする有形成分分類装置において、粒子に染色液中の色素を均一に結合させるようにすることで、実際の血球成分と同様な撮像画像が得られ、それにより分類装置のプログラムを何ら改変することなく、精度管理し、キャリブレーションを行なう試薬を提供できるため、体外診断用医薬品などの各種分析用途に利用することができ、産業界に寄与することが大である。 The present invention has a means for imaging a biological sample on a translucent plate or in a flow cell, and the means is equipped with an automatic focusing function for automatically focusing on a formed component in the biological sample. In the featured component classifier, by uniformly binding the dye in the staining solution to the particles, a captured image similar to the actual blood cell component can be obtained, thereby modifying the program of the classifier Therefore, it is possible to provide a reagent for accuracy control and calibration, which can be used for various analysis applications such as in-vitro diagnostic drugs and contribute greatly to the industry.
Claims (1)
（１）該粒子が、ポリスチレン、メラミン、ポリメタクリル酸およびガラスからなる群より選ばれた少なくとも１つ以上である。
（２）色素親和剤としてポリオキシエチレンオクチルフェニルエーテルを含む。 It has a means for imaging a biological sample in a translucent plate and is used for a formed component classification apparatus equipped with an automatic focusing function for automatically focusing on a formed component in the biological sample in the means. A standard reagent containing particles that further satisfies the following requirements (1) and (2):
(1) The particles are at least one selected from the group consisting of polystyrene, melamine, polymethacrylic acid and glass.
(2) Polyoxyethylene octyl phenyl ether is included as a dye affinity agent.
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| JP5783791B2 (en) * | 2010-05-11 | 2015-09-24 | 株式会社イヌイメデイックス | Staining solution for cleaning evaluation of medical equipment |
| JP6311698B2 (en) * | 2013-03-08 | 2018-04-18 | コニカミノルタ株式会社 | Staining for tissue staining, method for producing staining for tissue staining, and tissue staining kit including staining for tissue staining |
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