JP2000146782A - Apparatus and method for preparation of automatically fixed sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an apparatus and a method in which a high quality microscope sample for cell diagnosis is prepared from a cell suspension with high reproducibility and without manual intervention. SOLUTION: According to the presence or absence of continuity of an electrode 3, a level sensor 11 is turned on or off. The downstream side of a filter 1 is sucked by a vacuum pump 5 via a solenoid valve 10 and a constant pressure device 4. When the end point of a filtering operation is judged, the level sensor 11 is turned off, and the solenoid valve 10 is closed as so to stop the suction operation. A cleaning-liquid supply system 6, a first fixing-liquid supply system 7 and a second fixing-liquid supply system 8 are composed of tanks 61, 71, 81, of three-way selector valves 62, 72, 82 and of a syringe-type pump which is driven by a motor. By this constitution, under the control of a sequencer 9, an operator charges a sample in a prescribed amount into a sample container 2. By a simple depression of a start button, an immobilized cell sample is obtained on the filter 1 after about 20 minutes.

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【０００１】[0001]

【発明の属する技術】細胞を含む懸濁液、特に液状生体
試料中から細胞を濾過捕集するに当たって、その細胞を
純粋な形で、しかも変性させることなく捕集する必要が
ある場合に利用される。具体的には、尿中又は体くう液
等の液状検体に含まれる細胞の捕集、穿刺吸引物を生理
食塩水に分散させた試料からの細胞の捕集等に利用され
る。BACKGROUND OF THE INVENTION In filtering and collecting cells from a suspension containing cells, particularly from a liquid biological sample, it is used when the cells need to be collected in a pure form and without denaturation. You. Specifically, it is used for collection of cells contained in a liquid specimen such as urine or body fluid, collection of cells from a sample in which a puncture aspirate is dispersed in physiological saline, and the like.

【０００２】[0002]

【従来の技術】細胞懸濁液から細胞を濾過捕集する事は
従来から行われていたが、殆どは手動で吸引が行われ、
濾過の終了時点を肉眼で判断していた。このため吸引終
了時点の判断にばらつきがあり、吸引過度で細胞がフィ
ルターに食い込んだり、吸引不足で固定液が希釈される
等の変動があり再現性が得られなかった。2. Description of the Related Art Conventionally, filtration and collection of cells from a cell suspension have been performed, but most of them are manually suctioned.
The end point of the filtration was judged visually. For this reason, the determination at the end of aspiration was uneven, and there were fluctuations, such as cells invading the filter due to excessive aspiration, and dilution of the fixative due to insufficient aspiration, and reproducibility was not obtained.

【０００３】全自動固定標本作製装置としては、商品名
シンプレップ（ＴｈｉｎＰｒｅｐ）が販売されている。
技術内容の詳細は不明であるが、本発明との相違点は、
吸引を吸引速度一定で行っているため吸引圧力一定の保
証がないこと、濾過終了を吸引圧力の変化に依って判定
していること、及び生理的洗浄液による洗浄を行わない
ことにあり、高価な装置であり処理に時間がかかるとと
もに多検体自動処理に不向きである。[0003] As a fully automatic fixed specimen preparing apparatus, a product name "SinPrep" is sold.
Although the details of the technical content are unknown, the differences from the present invention are:
Since suction is performed at a constant suction speed, there is no guarantee of constant suction pressure, that the end of filtration is determined by a change in suction pressure, and that washing is not performed with a physiological washing solution, which is expensive. Since it is an apparatus, it takes a long time to process and is not suitable for multi-sample automatic processing.

【０００４】[0004]

【発明が解決しようとする課題】課題の一つはきれいな
顕微鏡観察のための試料が再現性良く得られること。二
番目は人手の節約、特に経験を要しないで誰でも容易に
良い試料が得られること。この２点が達成されれば標本
作製の標準化が可能となり、細胞診断そのものの標準化
が可能となる。One of the problems is that a clear sample for microscopic observation can be obtained with good reproducibility. The second is that it saves manpower, and anyone can easily obtain good samples without any experience. If these two points are achieved, standardization of specimen preparation becomes possible, and standardization of cytodiagnosis itself becomes possible.

【０００５】[0005]

【課題を解決するための手段】きれいな顕微鏡観察のた
めの試料を作るには一つには洗浄が有効である。フィル
ター上に残った夾雑物及び細胞の周囲に付いた微粒子を
洗浄により除去する事によって、きれいな資料が得られ
顕微鏡観察が容易になる。In order to prepare a sample for a clear microscope observation, washing is one of the effective methods. By removing the contaminants remaining on the filter and the fine particles attached to the periphery of the cells by washing, clear data can be obtained and observation with a microscope becomes easy.

【０００６】二番目には適当な吸引圧力を選定し、一定
に保つことが有効である。吸引圧力が大きすぎると細胞
がフィルターにめり込んで変形し、形状が変化して診断
に誤差が生じる。吸引速度一定では吸引圧力が一定にな
る保証がなく、早くフィルターに到着した細胞と後から
到着したものとの間に変形の差が生じる可能性がある。Secondly, it is effective to select an appropriate suction pressure and keep it constant. If the suction pressure is too high, the cells will sink into the filter and deform, resulting in a change in shape and an error in diagnosis. If the suction speed is constant, there is no guarantee that the suction pressure will be constant, and there is a possibility that a difference in deformation occurs between cells arriving at the filter early and cells arriving later.

【０００７】三番目には濾過終了の判定及び洗浄液、固
定液の投入のタイミングが重要である。濾過終了の判定
が遅いと空気を吸って細胞が乾燥し核の詳細な観察が不
能となる。判定が早過ぎると、洗浄液の場合は問題は無
いが、残留液状成分による固定液の希釈が起こり固定反
応の条件が十分満足されないため、細胞が部分的に固定
されるのでフィルター上に塗抹された細胞が剥離しやす
くなる。また、濾過終了の判定は良くても、洗浄液、固
定液の投入タイミングが遅れるとその間に細胞の乾燥が
おこり良好な試料が得られない。Thirdly, it is important to determine the end of filtration and to supply the washing solution and the fixing solution. If the determination of the end of filtration is too late, the cells are dried by inhaling the air and the nucleus cannot be observed in detail. If the judgment is too early, there is no problem in the case of the washing solution, but since the fixation solution is diluted by the residual liquid component and the conditions of the fixation reaction are not sufficiently satisfied, the cells are partially fixed, so that the cells are smeared on the filter. Cells are more likely to detach. In addition, even if the completion of filtration is good, if the timing of adding the washing solution and the fixing solution is delayed, the cells are dried during that time, and a good sample cannot be obtained.

【０００８】以上述べたように質の良い細胞診標本を再
現性良く作るには人手による操作を極力排除し、自動化
することが必要であり、また自動化によって省力化がは
かられるとともに、未熟練者でも、良質の試料が作成で
きるようになる。標本作製の標準化に当たっては多数の
試料を自動的に処理できる方式が必要となる。この点に
も考慮を払っておくことが必要である。As described above, in order to produce a high-quality cytological specimen with good reproducibility, it is necessary to eliminate the manual operation as much as possible and to automate the operation. Can produce high quality samples. Standardization of specimen preparation requires a method that can automatically process a large number of samples. It is necessary to take this point into consideration.

【０００９】濾過時には最適吸引圧力を一定に維持する
ことによりフィルター上での細胞の変形を防止し、濾過
終了時点を適切に判断し、洗浄液、固定液を注入する事
によって細胞の乾燥による劣化を防止し、濾過終了後に
洗浄液で洗浄する事によって細胞診標本の夾雑物を無く
して見やすくすることができる。一連の操作を自動化す
ることにより、これらの操作条件を再現性良く実施し、
高品質の細胞診標本を再現性良く作成することができ
る。At the time of filtration, the cell is prevented from being deformed on the filter by maintaining the optimum suction pressure at a constant level, and the end point of the filtration is appropriately determined, and the deterioration due to drying of the cells is performed by injecting the washing solution and the fixing solution. Prevention and washing with a washing solution after the completion of filtration can eliminate contaminants in the cytological specimen and make it easier to see. By automating a series of operations, these operation conditions are implemented with good reproducibility,
A high-quality cytological specimen can be prepared with good reproducibility.

【００１０】[0010]

【発明の実施の形態】尿を濾過して尿中に含まれる癌細
胞を捕集するための装置を図１に示す。他の目的の装置
も操作条件が異なるのみで、本質的には同一の装置が使
用される。この場合は血球を透過させ、癌細胞を捕集す
るためフィルター１の穴径は１０ミクロン、直径１２ミ
リメートルのものを使用した。尿サンプルは５０ｍｌを
サンプル容器２に投入する。本サンプル容器は下部に２
本の電極３１、３２を有し、３２の下端の位置が濾過の
終点判定にかかわる。この位置は実験により決定され
る。電極３１，３２間の導通の有無でレベルセンサー１
１がＯＮ／ＯＦＦする。フィルターの下流側は電磁弁１
０及び定圧装置４を経由して真空ポンプ５により吸引さ
れる。濾過の終点判定時にはレベルセンサー１１のＯＦ
Ｆで電磁弁１０が閉となり吸引を停止する。６は洗浄液
供給系、７、８は第一、第二固定液供給系を示す。これ
らはそれぞれタンク６１，７１，８１，三方切り替え弁
６１，６２，６３，モーターで駆動されるシリンジタイ
プのポンプから成る。９はこれらを制御するシーケンサ
ーを示す。DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows an apparatus for filtering urine and collecting cancer cells contained in the urine. Essentially the same device is used for other purpose devices, only the operating conditions are different. In this case, a filter 1 having a hole diameter of 10 μm and a diameter of 12 mm was used for transmitting blood cells and collecting cancer cells. 50 ml of the urine sample is put into the sample container 2. This sample container has two
It has the electrodes 31 and 32, and the position of the lower end of 32 is involved in the end point determination of the filtration. This position is determined by experiment. Level sensor 1 based on the presence or absence of conduction between electrodes 31 and 32
1 turns ON / OFF. Solenoid valve 1 downstream of the filter
The pressure is sucked by the vacuum pump 5 via the zero and the constant pressure device 4. When the end point of filtration is determined, the OF of the level sensor 11 is
At F, the solenoid valve 10 is closed to stop suction. Reference numeral 6 denotes a cleaning liquid supply system, and reference numerals 7 and 8 denote first and second fixing liquid supply systems. These are respectively composed of tanks 61, 71, 81, three-way switching valves 61, 62, 63, and a syringe type pump driven by a motor. Reference numeral 9 denotes a sequencer for controlling these.

【００１１】図２にサンプル容器２の詳細を示す。漏斗
状のサンプル容器にアルミ箔を接着して電極３１、３２
とした。３１はサンプル容器の下部まで、３２の下端は
３１より少し上になるよう接着した。FIG. 2 shows the details of the sample container 2. Electrodes 31 and 32 are bonded to a funnel-shaped sample container by bonding aluminum foil.
And 31 was adhered to the lower part of the sample container, and the lower end of 32 was slightly above 31.

【００１２】図３にシーケンサーの流れを示す。作業者
はサンプル容器２に所定量のサンプルを投入しスタート
ボタンを押すのみで、約２０分後にはフィルター１上に
固定された細胞の試料が得られる。この試料はすでに固
定を終わっており、急いで次の工程（スライドガラスへ
の転写、染色工程、等）に進める必要はなく、ほかの作
業が一段落した後に取り上げればよい。FIG. 3 shows the flow of the sequencer. The operator simply puts a predetermined amount of sample into the sample container 2 and presses the start button, and after about 20 minutes, a cell sample fixed on the filter 1 is obtained. Since this sample has already been fixed, it is not necessary to proceed to the next step (transfer to a slide glass, a dyeing step, etc.) in a hurry, and it is sufficient to take it up after the other work is completed.

【００１３】本装置につき各種条件を変更して実験の結
果、吸引圧力は水銀柱１０ｍｍ以下、電極３２の下端の
位置はサンプル容器の下端より１ｍｍが良く、洗浄液は
生理食塩水、生理的燐酸バッファー（ＰＢＳ）、１％Ｂ
ＳＡを含む生理食塩水等が好結果を示し、液量としては
５ｍｌで良かった。第一固定液はエタノール９５％水溶
液０．５−２ｍｌ、１５分間、第二固定液は２％カーボ
ワックスを含むイソプロピルアルコール、メタノール混
合水溶液０．５ｍｌ、反応時間２分間で好結果が得られ
た。As a result of an experiment under various conditions of this apparatus, the suction pressure is preferably 10 mm or less of mercury, the lower end of the electrode 32 is preferably 1 mm from the lower end of the sample container, and the washing liquid is physiological saline, physiological phosphate buffer ( PBS), 1% B
Physiological saline solution containing SA showed good results, and the liquid volume was 5 ml. Good results were obtained when the first fixing solution was 0.5-2 ml of a 95% aqueous ethanol solution for 15 minutes, and the second fixing solution was 0.5 ml of a mixed aqueous solution of isopropyl alcohol and methanol containing 2% carbowax and the reaction time was 2 minutes. .

【００１４】[0014]

【発明の効果】自動化により再現性良く良質の顕微鏡用
試料が得られる。一定の圧力で濾過を行うことによりフ
ィルターへの細胞の食い込み変形をなくす。濾過終了後
に洗浄液による洗浄を行うことにより夾雑物のないきれ
いな試料が得られる。濾過終了時点を電気的に判断して
吸引を停止し洗浄液を注入することにより細胞の変形と
劣化を防止し質の良い試料が得られる。According to the present invention, a high-quality microscope sample can be obtained with good reproducibility by automation. By performing the filtration at a constant pressure, the cells are prevented from biting and deforming into the filter. By performing washing with a washing solution after completion of the filtration, a clean sample free of impurities can be obtained. By electrically judging the end point of the filtration, stopping the suction and injecting the washing solution, the deformation and deterioration of the cells are prevented, and a high quality sample is obtained.

【図面の簡単な説明】[Brief description of the drawings]

【図１】本発明の装置の構成を示す。FIG. 1 shows the configuration of the apparatus of the present invention.

【図２】サンプル容器とそれに取り付けた電極を示す。FIG. 2 shows a sample container and electrodes attached thereto.

【図３】本装置の一連の操作の流れを示す。FIG. 3 shows a flow of a series of operations of the present apparatus.

【符号の説明】[Explanation of symbols]

１ フィルター ２ サンプル容器 ３ 電極 ４ 定圧装置 ５ 真空ポンプ ６ 洗浄液供給系 ７ 第一固定液供給系 ８ 第二固定液供給系 ９ シーケンサー １０ 電磁弁 １１ レベルセンサー DESCRIPTION OF SYMBOLS 1 Filter 2 Sample container 3 Electrode 4 Constant pressure device 5 Vacuum pump 6 Cleaning liquid supply system 7 First fixed liquid supply system 8 Second fixed liquid supply system 9 Sequencer 10 Solenoid valve 11 Level sensor

───────────────────────────────────────────────────── フロントページの続き (72)発明者 河合 義雄 東京都武蔵野市吉祥寺東町３−12−10 Ｆターム(参考） 4B029 AA07 AA27 BB01 BB11 CC01 FA01 FA11 FA15  ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshio Kawai 3-12-10 Kichijoji Higashicho, Musashino City, Tokyo F-term (reference) 4B029 AA07 AA27 BB01 BB11 CC01 FA01 FA11 FA15

Claims (2)

【特許請求の範囲】[Claims] 【請求項１】 生体試料からなる細胞浮遊液を濾過して
細胞を捕集し細胞診断用プレパラートを作成する一連の
操作の前半の部分、具体的には細胞浮遊液から細胞を濾
過捕集しフィルター上で細胞の固定を行うところまでの
操作、を自動的に行うための装置であり、一定の吸引圧
力による細胞浮遊液の吸引濾過、濾過終了を自動的に判
定して吸引停止と所定量の洗浄液の自動投入、洗浄液の
濾過終了を自動的に判定しての所定量の第一固定液の自
動投入と第一固定化反応時間の管理、及び必要に応じて
の、第二固定液の自動投入と第二固定化反応時間の管理
を自動的に行い、フィルター上に細胞を変性させること
なく捕集し固定することを特徴とする自動固定標本作製
装置。1. A first part of a series of operations for preparing a cell diagnostic preparation by filtering a cell suspension comprising a biological sample to collect cells, specifically, filtering and collecting cells from the cell suspension. This is a device for automatically performing the operation up to the point where the cells are fixed on the filter.The suction filtration of the cell suspension with a constant suction pressure, the end of the filtration is automatically determined, and the suction is stopped and the predetermined amount is stopped. Automatic injection of the washing solution, automatic injection of a predetermined amount of the first fixing solution by automatically determining the end of the filtration of the washing solution and management of the first fixing reaction time, and, if necessary, the second fixing solution An automatic fixed sample preparation apparatus characterized in that automatic injection and control of a second immobilization reaction time are automatically performed, and cells are collected and fixed on a filter without denaturation. 【請求項２】 生体試料からなる細胞浮遊液を濾過して
細胞を捕集し細胞診断用プレパラートを作成する一連の
操作の前半の部分、具体的には細胞浮遊液から細胞を濾
過捕集しフィルター上で細胞の固定を行うところまでの
操作、に於いて、細胞浮遊液を濾過した後にフィルター
上に残った細胞を生理的洗浄液、例えば生理食塩水、燐
酸バッファー、１％ＢＳＡ含有生理食塩水等、で洗浄し
付着粒子等を除去すること、及び濾過が終了した直後に
生理的洗浄液を添加し、空気を吸引する事により細胞が
乾燥変質する事を防止することを特徴とする細胞診断用
プレパラート作成前処理方法。2. The first half of a series of operations for collecting cells by filtering a cell suspension comprising a biological sample to prepare a cell diagnostic preparation, specifically, filtering and collecting cells from the cell suspension. In the operation up to the step of fixing the cells on the filter, the cells remaining on the filter after filtering the cell suspension are washed with a physiological washing solution, for example, a physiological saline solution, a phosphate buffer, and a physiological saline solution containing 1% BSA. For cell diagnostics, washing is performed to remove adhered particles and the like, and a physiological washing solution is added immediately after the filtration is completed, and the air is sucked to prevent cells from drying and altering. Preparatory preparation preparation method.