EP1574259A1 - A method and apparatus for detecting of marked microparticles - Google Patents

Abstract

The device detects marked microparticles in a conductive medium (4), in particular extra-corporal blood, where micro particles have been introduced with magnetically activated marked parts and one detectable fluorescent marking. The microparticles collect in the flowing medium with the aid of a magnetic field. They are continuously detected at the deposit point (21) on the inside wall of the circuit due to the additional marking. The conduction circuit with transparent wall serves to conduct the medium flow. It has at least one magnet (19) to produce the magnetic field in the circuit and a fluorescence detector (18) whose rays penetrate through the wall to the potential deposit area of microparticles inside.

Description

The invention relates to a method for the detection of labeled microparticles in a medium flowing through a conduit

Likewise, the invention relates to a device for the detection of magnetic and fluorescently labeled microparticles in a flowing medium

There are certain blood purification procedures, such as plasmapheresis and hemoperfusion / plasma perfusion, which detoxify by removing more protein bound or more hydrophobic Substances from the blood takes place. Unfortunately, the effectiveness of these procedures is common limited by technical problems, low selectivity and low performance. On the other hand, symptoms of patients suffering from liver failure or other hepatic dysfunction, with the elimination of toxins and others not desired substances, which are treated with conventional dialysis treatments (hemodialysis, Hemofiltration) can not be eliminated. In Fig. 1 is a for the shown suitable system, also known from EP 0 776 223 B1 has become. In this system or device, a primary extracorporeal circuit 3 is attached to one Patient PAT connected to an arterial line 1 via a blood pump 2 too a filter 5, from which the return flow via a venous line 4 to the patient PAT is done. The second extracorporeal circuit or secondary circuit 7 leads from the filtrate side of the filter 5 via a centrifugal or roller pump 8 back to the filtrate side of the Plasma filter 5. In the primary circuit, moreover, a device 6 for detecting certain Properties of the filtrate flow included. The device shown in Fig. 1 is state of Technique, wherein the invention with the device 6 explained in more detail below and an associated measurement method.

Returning to Fig. 1, it should be explained that by the relative movement of the liquids of primary and secondary circuit a transmembrane pressure in the filter 5 is formed, the results in a fluid exchange between the circuits result. In the secondary circuit 7 circulate microparticles 9, with the help of which toxins specifically bound and so from the Blood are removed. Such microparticles or microspheres have a diameter of less than 20 .mu.m, in particular 1 to 7 .mu.m, which corresponds to the diameter of the Blood cells is comparable. Special characteristics of microspheres are a big one outer surface and short diffusion paths to inner pores, if any.

To prevent direct contact between the blood cells and the particles is the whole blood with the filter in a cell portion and a filtrate, z. As plasma, wherein the filtrate circulates in the secondary circuit at high speed to a high Filtrattransmembranfluss to be able to sustain for a powerful treatment. The Flow rate in the secondary circuit 7, which is typically 0.5 to 4 l / min, is also so high, so that no formation of deposits can take place.

In order to keep patient safety as high as possible, the implementation is different Security systems in a system as shown in Fig. 1, required. The Microparticle suspension of secondary circuit 7 is only from the primary blood circulation separated by a thin-walled hollow fiber membrane. In the case of a diaphragm rupture or even a single leak would result in an infusion of microparticles into the patient's bloodstream. To prevent this, the device 6 is arranged to prevent the occurrence of Detecting microparticles in the primary circuit 3. Be microparticles in the Blood circulation detected, can take immediate action, such as switching off the blood pump 2 etc., taken or triggered automatically.

The already mentioned EP 0 776 228 B1 also addresses the problem of the detection of microparticles in the bloodstream and suggests ultrasonic sensors as well as optical Sensors, in particular in connection with a coloring of the liquid in Sekudärkreislauf with, for example, fluorescent substances. On the other hand, it is also on it pointed out that one microparticles, which may have arrived in the primary circuit, with the help of a magnetic field, if the particles are magnetically activated Shares included. Such separation of magnetically marked particles, in particular Cells in a high gradient field are also described, for example, in EP 1 019 195 B1 described.

A known device for measuring optical properties in the primary (blood) circle 3 is shown in Fig. 2. To distinguish the microparticles from the surrounding blood components To be able to, they are with an optically reflective or preferably with labeled with a fluorescent dye. Such labeled particles become the secondary circuit 7, in addition to the (adsorber) microparticles contained in it. typically, 1 to 10% V / V labeled particles are added to the adsorber particles and the total particle concentration is about 20% V / V. The labeled particles circulate then together with the Adsorberpartikeln in the secondary circuit 7. In the case of Filter leakage occur, the labeled particles together with the adsorbent in the Primary circuit 3 via.

With the aid of the detection device 6, an occurrence of fluorescently labeled particles in the primary circuit 3 are also recorded quantitatively and it can be calculated from the amount of Indicator particles on the amount of in the adsorber particles in the primary circuit passed Adsorberpartikel be closed. It is understood that the size distribution the marked particle should be equal to that of the adsorber particles so that this inference sure enough.

FIG. 2 also shows the optical principle of a known fluorescence-marked detection device 6 Microparticles. A light source 10, z. As an LED or a laser, provides the Excitation light, in the example shown in the range of 57o to 610 nm. The beam of Light source 10 is focused and / or corrected by means of a lens 11 and then with the help of optical filter 12 from the broadband light, the dominant excitation wavelength of the fluorescent dye, here 590 nm, filtered out.

A semitransparent 45 ° beam splitter 13 directs the excitation light to a focusing lens 14 to focus the beam on the liquid volume to be observed, which in the venous line 4 of the primary circuit 3 is located. This line 4 is at least partially and in a known manner z. B. formed as a transparent tube whose material is chosen is that it in the range of excitation and fluorescence wavelength to no optical Impairment for the detector with respect to absorption or autofluorescence results. Of course, in a particular, located in the primary circuit 3 measuring chamber instead of being measured in a tube section.

Fluorescent light emitted by labeled microparticles is removed by means of the lens 14 bundled and passes through the beam splitter 13 in the receiving path. From one emitted Spectrum from 610 to 635 nm or any parasitic radiation from the excitation path and ambient light by means of optical filters 15 light of a certain wavelength, filtered out in the present case 620 nm and a focusing lens 16 to a Photodetector 17 out, in a practical design z. B. on the active surface a photomultiplier. The electrical output of the photodetector 17 is proportional the light intensity and also from the signal amplitude can affect the density of fluorescently labeled Particles are closed in the considered liquid volume. Of the shown fluorescence detector is designated here and in further figures with 18.

A problem specific to the prior art is the relatively low sensitivity the detection method. Thus, for fluorescence light detection, the resulting Signal-to-noise ratio due to the high optical absorption of blood at the extraction and emission wavelength of conventional fluorescent dyes a limiting Factor, because it can only ever due to the high optical density of the blood small liquid volume are detected by the detector. The focus of the detector beam path is only on a thin layer within a blood transporting one Hose od. Like. Directed and there are accordingly few of any existing Particles detected. However, the intensity of the measured fluorescent light is the Particle number in the considered volume proportional.

It should be noted at this point that the use of magnetically marked particles in analytical methods is known. Thus, WO 92/14138 shows an examination method including an associated device in which prepares small-volume samples which contain a complex with magnetic particles bound thereto. This sample is then placed in a small sample chamber and there are the Complex particles by means of a magnetic field to a disposed within the chamber Electrode pulled to then, after applying a voltage, an electrochemiluminescence stimulate, which is then detected. This procedure uses electrodes within a special measuring chamber ahead and works discontinuously.

JP 9089774 A describes a fluorescence microscope with an annular permanent magnet at the objective lens for concentration of magnetic and fluorescent particles in front of the lens, where again the measurement is discontinuous and in a measuring chamber with a small, defined volume.

JP 5264547 discloses an immuno-technological method in which for analysis a sample is labeled enzymatically and magnetically Sample substance is drawn onto a substrate with which it reacts to then analyzed become. Only the reaction with the substrate allows the detection by an absorption or fluorescence analysis.

It is an object of the invention to provide a method and a device, with their aid labeled, in particular fluorescence-labeled microparticles in a flowing Medium such. B. in a bloodstream, already reliably detected in low concentration can be.

This object is achieved by a method of the type mentioned, in which According to the invention, microparticles are used which, on the one hand, are magnetically activatable On the other hand, at least one other detectable Have marker, and the microparticles using a magnetic field in the flowing Medium captured and accumulated and stored at the collection point as a deposit at the Inner wall of the line continuously detected based on their further mark become.

Likewise, the object is achieved with a device of the type indicated above, which Is characterized by a line with an optically transparent Wall for guiding the medium flow, at least one magnet for generating a magnetic field is arranged in the interior of the conduit on the outside thereof, wherein the Magnetic field in the presence of the labeled microparticles to a deposition of Particles on the inner wall of the conduit leads, and a fluorescence detector whose Beam path through the wall of the line in the interior to the area of a possible deposition of microparticles.

In a particularly practicable variant, it is provided that the further marking an optical fluorescent label and the microparticles at the collection site optically be detected.

The invention is particularly suitable for a blood purification system whose extracorporeal Blood flow forms the flowing medium to increase safety for the patient.

It is expedient if the medium in a line with optically permeable Wall flows and the magnetic field with the help of at least one outside the line generated magnet is generated. In this case, a conventional, used in the infusion technique Hose serve as a conduit and there are no sterilization problems. there is particularly suitable for an embodiment in which in the region of the at least one magnet the deposition of trapped and labeled microparticles on the inner wall the line is detected by means of the beam path of a fluorescence detector.

To simplify the device, it is expedient if the at least one magnet a permanent magnet.

A well-detectable capture of microparticles results when a magnet near the line, is arranged immediately upstream of the entrance of the beam path in the line.

It results in a compact construction when a magnet is located near the conduit, the north-south axis of the magnet being at an angle to the beam path of the magnet Fluorescence detector, but essentially in a common plane normal to Line is. The angle between north-south direction of the magnet and Beam path of the detector expediently between 70 ° and 100 °.

When two magnets are provided, which are arranged in a normal plane to the line are, with the north-south axes of the magnets below 60 ° to 120 °, preferably 90 ° are inclined towards each other and the beam path of the detector in the direction of the line seen essentially by the angular symmetry of the north-south axes, receives a particularly effective capture of the microparticles, where it is the best possible Optical detection of the deposit may be advisable if the beam path of the fluorescence detector slightly downstream of the axes of the magnets through the wall of the Line runs.

Fig. 1

eine Vorrichtung nach dem Stand der Technik zur Beseitigung von Giftstoffen aus Blut,

Fig. 2

eine bekannte Vorrichtung zur Messung optischer Eigenschaften in dem Blutkreislauf der Vorrichtung nach Fig. 1,

Fig. 3

eine schematische Detailansicht einer ersten Ausführungsform der Erfindung, normal zur Flussrichtung gesehen,

Fig. 4

in einer schematischen Detailansicht in Flussrichtung gesehen eine Variante der Erfindung,

Fig. 5

in einer Darstellung wie Fig. 3 eine weitere Ausführungsform der Erfindung,

Fig. 6

die Ausführung nach Fig. 5 in Flussrichtung gesehen und

Fig. 7

in einem Diagramm gemessene Ausgangssignale des Detektors einer Vorrichtung nach der Erfindung.

The invention together with further features is explained in more detail below by way of example embodiments, which are illustrated in the drawing. In this show

Fig. 1

a device according to the prior art for the removal of toxins from blood,

Fig. 2

a known device for measuring optical properties in the blood circulation of the device according to Fig. 1,

Fig. 3

1 is a schematic detail view of a first embodiment of the invention, seen normal to the flow direction,

Fig. 4

in a schematic detail view in the direction of flow, a variant of the invention,

Fig. 5

in a representation like FIG. 3 shows a further embodiment of the invention,

Fig. 6

the embodiment of FIG. 5 seen in the flow direction and

Fig. 7

in a diagram measured output signals of the detector of a device according to the invention.

To the sensitivity of known devices and systems for the detection of marked To increase microparticles, the invention provides, in addition to a first mark - in the shown embodiments a fluorescent label - another marker, namely a magnetic marker to use. Here are eligible microparticles own z. For example, a microspherical cellulosic matrix having an iron (II, III) oxide core. For fluorescence labeling, the dye cresyl violet (9-diamino-benzophenoxazonium perchlorate) with 1,4-glycidyloxypropyltrimethoxylsilane on the particle surface be fixed, with a preferred diameter of the thus marked Particles between 5 and 15 microns. Of course, the mark to be detected could also another kind, z. As a radioactive, but has the fluorescent label in the Practice as particularly suitable as cheap, safe and with reasonable effort detectable, exposed.

As is apparent from Fig. 3, the invention further provides the microparticles to be detected using a magnetic field to capture and collect them to the collection point to detect on the basis of their fluorescent label. In the embodiment shown is in the vicinity of a tube portion of the venous line 4 is a permanent magnet 19 arranged. In the flow direction downstream of the magnet 19 is the tip of the Fluorescence detector 18, which is shown in Fig. 2 and of which the lens 14 and the can be seen through this bundled beam path 20.

If the magnetic field of the magnet 19 is strong enough - the magnet 19 or the magnets Of course, these and the other embodiments can also be used as electromagnets be formed - remain all magnetically marked particles in the region of the magnet 19 adhere to the inner hose wall. This deposit of 21 labeled particles becomes pulled along by the flow of part of the magnet 19 downstream. In the practical One realizes that there is a significant area of deposit 21 in the Focus of the beam path 20 is located.

The fluorescence detector 18 measures the intensity of the fluorescence radiation emitted by the starting with the excitation wavelength irradiated microparticles. As the marked Collect particles at the collection point or magnetic trap and not - as in the stand The technology - to be measured in the flow past, can practically the entire Amount of the marked in the bloodstream 3 tagged particles are determined. Measurements have shown that the intensity of the fluorescent light is substantially proportional to the total amount of the transferred into the primary circuit 3 particles.

In the variant according to FIG. 4, two magnets 19a, 19b are aligned radially and around one Angle of z. B. 60 ° to 120 °, preferably 90 ° offset from each other with respect to the (Venous) line 4 is arranged. In this case, the beam path 20 of the detector 18 expediently by the angular symmetry of the magnets 19a, 19b. As the marked Particles between the two magnets 19a, 19b deposit, the deposits are in one free optical path for the detector 18 and the particles can be directly at the collection point be irradiated with the excitation light. Conveniently, the beam path 20 of the fluorescence detector 18 slightly downstream of the axes of the magnets 19a, 19b the wall of the conduit 4 run to the "smearing" of the deposit 19 (see Fig. 2) due to the flow to correct.

A third variant with respect to the relative position of the beam path of the detector 19, magnet 19 and line 4 is shown in FIGS. 5 and 6. Here lies the north-south magnetic axis of a Permanent magnet 19 at an angle of 60 ° to 120 °, preferably normal with respect the beam path 20 of the detector 18 and also with respect to the axis of the line 4. Magnetic labeled microparticles accumulate on the magnet 19 facing inside the line 4 and are illuminated laterally by the detector beam. The lateral position The detector beam is adjusted during operation so that a maximum output signal results.

The measurement results shown in FIG. 7 clearly illustrate the achievable with the invention Improve sensitivity and increase signal-to-noise ratio. The Line 22 shows the output of photodetector 17 in the absence of fluorescently labeled Particles, thus the background noise. On the ordinate is the height of the output signal plotted on the abscissa, the particle volume, more precisely the particle volume, which has already passed the measuring point.

The line 23 relates to a measurement with fluorescence-labeled and also ferromagnetic marked particles in an arrangement such. B. of Fig. 3 with the magnet removed 19. Es results in a detectable, but only slightly above the noise floor Output signal, regardless of the total flowed past the measuring point Particle volume is. A similar line results when the particles are only fluorescently labeled are, whether a magnet 19 is present or not.

Finally, the curve 24 shows a measurement result when using the invention, wherein can be seen clearly the increase of the signal / noise ratio, which on an average Doubling of the output signal is due. At the measuring point is in the ordinate direction, the standard deviation of the output signals for five measurements located. The marked particles are captured by the magnet and held. The number of retained particles increases with the total amount of the magnetic trap entered particles and causes an increase in fluorescence intensity.

Claims (12)

Method for the detection of labeled microparticles in a medium flowing through a conduit,
characterized in that
Microparticles are used, which on the one hand have magnetically activatable marker portions and on the other hand have at least one detectable marker, and the microparticles trapped by means of a magnetic field in the flowing medium and accumulated at the collection point as a deposit (21) on the inner wall of the line based their further mark are detected continuously. A method according to claim 1, characterized in that the further label is an optical fluorescent label and the microparticles are optically detected at the collection point. A method according to claim 1 or 2, characterized in that the flowing medium is an extracorporeal blood flow of a blood purification system. Method according to one of claims 1 to 3, characterized in that the medium flows in a line (4) with optically permeable wall and the magnetic field by means of at least one outside of the line (4) located magnet (19; 19a, 19b) is generated , A method according to claim 2 and 4, characterized in that in the region of the at least one magnet (19; 19, 19b) the deposition (21) of trapped and marked microparticles on the inner wall of the conduit (4) by means of the beam path (20) of a Fluorescence detector (18) is detected. Device for the detection of magnetically and fluorescently labeled microparticles in a flowing medium
marked by
a line (4) with an optically permeable wall for guiding the medium flow,
at least one magnet (19; 19a, 19b) arranged to generate a magnetic field inside the conduit (4) on the outside thereof, wherein the magnetic field in the presence of the labeled microparticles to a deposition (21) of the particles on the inner wall of the conduit leads, and
a fluorescence detector (18) whose beam path (20) extends through the wall of the conduit (4) in its interior to the region of a possible deposit (21) of microparticles. Apparatus according to claim 6, characterized in that the at least one magnet (19; 19a, 19b) is a permanent magnet. Apparatus according to claim 6 or 7, characterized in that the magnet (19) near the conduit (4), immediately upstream of the entrance of the beam path (20) is arranged in the conduit. Apparatus according to claim 6 or 7, characterized in that the magnet (19) is located near the conduit (4), wherein the north-south axis of the magnet (19) at an angle to the beam path (20) of the fluorescence detector (18 ), but substantially in a common normal plane to the line (4). Apparatus according to claim 9, characterized in that the angle between the north-south direction of the magnet (19) and the beam path (20) of the detector (18) is between 60 ° and 120 °. Apparatus according to claim 7, characterized in that the two magnets (19a, 19b) are arranged in a plane normal to the conduit (4), wherein the north-south axes of the magnets (19a, 19b) below 60 ° to 120 °, preferably 90 °, are mutually inclined and the beam path (20) of the detector (18) seen in the direction of the line substantially through the angular symmetry of the north-south axes. Apparatus according to claim 11, characterized in that the beam path (20) of the fluorescence detector (18) extends slightly downstream of the axes of the magnets (19a, 19b) through the wall of the conduit (4).

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