DD222827A5 - Apparatus and method for cell selection - Google Patents

Abstract

Apparatus and method for selecting specific biological cells from other cells and observing at least one selected property of the selected biological cells, characterized by traeger means having a predetermined distance between an upper surface that can be fed with biological cells and an opposing floor surface, with a Variety of recesses, which extend in a predetermined order between the two surfaces. The recesses have a predetermined shape and given dimensions on the upper and the Bodenflaeche, which are defined as upper and bottom dimensions accordingly. When loading the upper surface with biological cells, only selected cells of predetermined dimensions are essentially kept in the recesses, essentially only one cell per hole. Fig. 16

Description

Berlin, 24.08.1984 ¹ . - 62 424/17

Device and method sur cell selection

Field of application of the invention

The present invention relates to a device, and a method for cell selection and. especially on

, those for "keeping" single cells in known locations, thereby distinguishing, among other ways of selection, at least one subpopulation of cells using defined parameters common to all members of the subpopulation.

can isolate from a more general cell population,

'. Characteristic of the known technical solutions

Devices and methods for reading and separating subpopulations of biological cells, such as the ropes contained in the blood, are already known. Several of the methods considered to be important and considered to be characteristic of the state of the art are briefly described and commented on below »

Cell adhesion-based separation, This method is not very efficient and is not suitable for separating different cells having the same membrane properties as, for example, B cells adhering to glass.

Iinraunfluoressenz-separation, this method is based 'on' known binding properties of cell-Subpooulationen to certain antigens and / or antibodies ran binding to the cells in a later stage can be used to · identifying the ropes. However, this method is of rather limited value, in that it is not _: used for.-Caim. To separate cells on the basis of measurements of their biological activities or their reaction to the bound substance,

, .... '' -2-

Electrophoresis. This method is based on the separation of cells depending on the electrical charge of the cell. Thus, it can not be applied to separate different groups of cells having the same charge or mass.

i '. ·'

Radioassav including radioimmunoassay, radio inkorpora- '. tionassay, radioehsy Biological Assay, With this. Method, it is neither possible to separate cell groups from each other, nor is it possible to distinguish within a group a subgroup based on their activity or inactivity and response β>.

Morphology, The distinction between cells based on. their physical appearance. This method is: to handle rasQh, but is also the coarsest of all _β

Tissue separation according to the specific gravity (gradient technique). In this procedure the cells float on an isotonic solution of known density, viscosity and viscosity. This configuration is obtained by centrifuging acceleration forces at a certain temperature / and

. '' s-sedition exposed »cells, whose specific mass grcs-.ser than that of the solution, are sinking. Cells with the same specific density become the solution and are suspended in it, and cells with a specific density lower than the solution float on their surface. The main problem of this method is the Xompart imentation of the cells within the density gradient by 'ümgebuiigsbedln-'. "such as 'temperature,' osmolarity, acceleration, • 2, 3, the distance of the interface between the blood and the gradient from the axis of rotation be influenced.

In addition to the above shortcomings of the various previous methods, there is a considerable drawback to the fact that the separated cells almost always contain cells which do not belong to the group or subgroup of interest. Thus, any diagnosis of the cells separated by any of these methods is necessarily inaccurate, even if all the procedures have been performed with the greatest precision.

L, Gereck et al. describe, for example, an SCM test (jSucturedness of JGjyt © plastic, .Matrix) in the Biophys. !., July 1978, J hg. 23, STr. 1, p. 395 ff; In the abovementioned article, the authors state that cells which have been separated by the abovementioned gradient method, despite the greatest care during the test procedure, for example. 50, - contained unwanted cells ».

The inability to completely segregate a particular cell group from all other groups impairs diagnostic accuracy, in high measure. In addition, the most significant factor is the fact that, in the methods described above, cell deposition and subsequent tests on the ropes are more likely to be on a macro- ¹ or batch basis than on a ilikrc basis, with ilikrc . Based on the one considered, in which the read cells are separated from each other and in which each one cell can be tested and examined separately. Any. System bsw. One method of separating rejected cells of interest from other cells and further separating the read-out cells from each other so that they can be tested and / or examined would be of great importance in the diagnosis of various biological conditions ) O-VJ-S OG - the ϊβΓυι ^^ ο cU ^ Sicr iiViC ^. ^ 0 ats-ü. aJ <J, S ilu "

Examining individual readout cells would eliminate errors that currently exist in numerous diagnoses made on the basis of inexact statistical criteria.

Aim of the invention; , ,

The aim of the invention is to overcome the inaccuracies in the known methods,

Explanation of the essence of the invention '.. _. ,

It is therefore the object of the present invention to provide a method and equipment for reading out a 'row group' from other rows, as well as for separating the a.usgelese aen. Cells from each other. Each cell read and separated from each other is located in a well-known location, all of the cells that have been read out can be subjected to the usual tests, and the effect on each individual cell can be best assessed, which in turn allows a more accurate diagnosis. ; IRD. The tests and the effects on each cell can be automatic. · To be reduced to reduce the impact - a task that. may also be carried out by relatively unskilled personnel,

This object is achieved by an apparatus for selecting speaf biological cells from other cells and observing at least one selected property of the selected biological cells, which is characterized by an array of support means having a precursor The distance between the surface and the upper surface, which is chargeable with the biological cells _? ¹ year of a

Overlying floor surface, having a plurality of finishes extending in predetermined order between both surfaces, the recesses having a predetermined configuration with predetermined dimensions at the top and bottom surfaces, which are correspondingly definable as top and bottom dimensions, wherein when feeding the upper surface with biological cells only selected cells of predetermined dimensions are kept substantially in the recesses with. 7 / one cell, "per hole."

For observing at least one selected property of selected biological cells, in the carrier means the bottom dimension of each recess is greater than the smallest dimension of the internal cross section. ,

The upper bottom dimension of each recess lies in the micrometer range «

It is advantageous if the thickness of the support means and the upper dimensions of the recesses are of the order of the diameters of the selected cells.

Suitably, the recesses are dimensioned so that they can accommodate lymphocytes, and sv / ar a recess per «

The assembly also has first structural features for supporting the support means at a particular location thereof, each recess, the support, being in a known location definable as a "particular location" with respect to the structural members, as well as Instruments for the observation and / or measurement of at least one cell characteristic and control devices for the control of the instruments, for any instrument

save, based on their specific location, to be able to judge and measure at least one property of the cell in it. , , , ~

The instruments include advantageously optical detection instruments for determining optical properties of any of the cells,

The first, structural elements include means for supporting a plurality of spatially separated support means, each having cells in their recesses, and second structural members. which are adjustable with respect to the first structural elements so that the cells on the spatially separated support means can be stimulated equilibrium by a given substance, the control means being able to direct the instruments to the particular location of any of the recesses on any one to direct the means of support to observe the cell inside it »··

The first and second structural elements are mutually adjustable and the cells can be stimulated either from the top or from the bottom of the recesses. "By moistening they become". ':

The zvieiteh structural elements include, such. which contain mutually different stimuli, the first and second structural elements being mutually adjustable and stimulating cells in at least some of the spatially separated carriers simultaneously by means of the different stimuli means; become * j · '

-T-

Among the means of support are the force generating sines which direct any of the cells to the recess in which they are. are arranged, holds or pushes out of this. ·

Among the force-generating Sinrichtungen include those that were based on the recesses in predetermined profiles ¹ to hold any of the cells to the recess in which it is arranged by means of electromagnetic devices su or they herauszubefördern out of the recess.

The force-generating means may also include those which produce a pressure differential across at least a selected part of the carrier means or those which increase the adhesiveness of the cells in the notches.

3s may be advantageous, 7. ^r hen -to the first structural elements such sum supporting a plurality of spaced-apart support means are each \ Zeil contain en in their recesses, and -second structural elements which are selectively adjustable with the first structural elements in order to allow the cells on the spatially separated support means to be stimulated equilibrium with given origin, with control means capable of directing the instruments to the particular crt of any of the recesses or any of the support means observe cell in it «

It is expedient that .the first and second structural elements ¹ against each other bs; v · to each other are adjustable, wherein the cells are stimulated with either from the top or from the bottom of the savings by moistening.

The second structural elements include devices having different stimulating substances therein, and the first and second structural elements are mutually adjustable and the two parts in at least some of the spatially separated support devices are stimulated at the same time by the different stimulating substances , ·,,

The method for separating at least one specific biological cell group to facilitate the observation of selected cells in the separated group is characterized in that the top of a support having a predetermined thickness between the top and the opposite base has Cells are covered, wherein the carrier has recesses in predetermined order, which lie between the said-pages, and that at substantially 'complete coverage' of the carrier by cells only selected 'cells, based on their dimensions, substantially within the recesses are held, wherein essentially one cell per recess is given hur.

Furthermore, the top of the carrier is washed to remove all '-'. not deposited in the recesses en.Zellen -zu remove, with essentially only the severed lines in the ^! Carrier constructions remain with essentially one cell per recess »:.

It is expedient, furthermore, that as a puncture of a known cell property, any cell is held or pushed out of the recess in which it is deposited. ,

Advantageously, said support means is mounted in predetermined orientation on a support structure, the position, recess and row therein being definable, as the location of the cell is known to facilitate control of instruments to each of said recesses each one is observed and determined by a variety of cell characteristics, including those properties resulting from its stimulation by the predetermined substance,.

The cells on the carrier, after prior stimulation, are treated to return to their previous state. -

Furthermore, the following steps are expedient: neutralization of the ropes on the carrier after a previous stimulation and at least stimulation of the cells by different substances.

It is advantageous if, in addition, the cells which have been separated are increased in order to obtain a higher number of separated cells, it being possible for the cells which are propagated to be the cells of "the Carrier devices are removed, or that the cells that are propagated, - are separated cells that remain on the Trägerein-, • directions »

rs r> T.

It may also be advantageous to avoid the cells while maintaining their positions in their respective recesses,

, ι

It may also be expedient that further at least one cell, which is not among the separated cells, is killed while the row is in place, namely in a:

, , -10- .. '.

Recess of the carrier. '.

*

Furthermore, the proliferation of living cells on the. Carrier on the spot "".

Ss can also be .from advantage that the further ceremony Vera operation of at least one cell which multiplies in-situ 'beobachtet'wird ·'.

embodiment

The present invention will be described below with reference to various. Examples of B-rfinalrig- and 'der. attached drawings are explained in detail. Show it:

Mg »1Α..φ10: Scheaiyan bar positions - partly in the transverse. . '. cut - of preferred erfindungsgeciäßen .. cell carriers}

Fig. ' 2A _e . ₄ 2D; an example of a multi-axis cell carrier.

for the execution of measuring cycles for a large number of cell carriers j

.Pig, 3A U.3B: enlarged sectional views of the holder

4: the Gchiatic representation of an optical

Analyzer sum individual scanning of the cells of the population contained in a cell carrier. "

5: schematically a white example of an optical analyzer. , '

Figs. 6 and 6A ... 6C: modified holders of the examples according to Figs

5 ¹ Ig. 3;

FIGS. 7A and 7B:

a possible form of a flow chamber for the bottom side of an analysis system;

Fig. Si

a modified version of the flow chamber of fig. 75

Fig. 9:

a further possible embodiment of a holder and a Durchflußkamrrier for a further developed analysis system;

Fig. 10:

a separate ion unit adapted to receive the holder of Fig. 6 for supplying the cell carriers with cells;

Fig. 11:

a sectional view of parts of the embodiment shown in Figure 10;

Figs. 12A and 12B: one for receiving the holder of Fig. 9;

adapted separation unit for supplying the cell carriers with cells;

Fig. 13A ... 13C:

Details of an initialization element, dps, for cie1 sinsets were adapted in the ris.cr.ien of the ° seo-sessional unit according to FIG. 12;

Fig. 14

a cross-section of another

Embodiment of an -iiupply-

supply

sisG '^; exites which has been adapted with the separation unit of Fig. 12;

FIGS. 15A and 15B show an embodiment of a multiple carrier.

_v systems for clinical use, wherein

, the separation and measurement steps.

· '. ", are summarized;

Fig. 16 · i ^": · ;, a -sch.em.at is before Gesatnt illustration of a ';: ·' e.rfindungsgemäßen optical Diagnosesystenis;

Pig. -17: a cell carrier for selective attraction.

hen or release of desired cells,

The invention is first of all limited in a manner limited to: selecting and analyzing a particular cell population; certain over-.anderen Zeil- populat ion ^'1 in einer'biologischen liquid contained type described * In addition, a further selection of a special sub-population can be separated from the each read population. Specifically, the invention will be described first in connection with selecting and analyzing a particular subpopulation of lymphocytes present in human blood, the lyciphocytes being first separated from other strands, and then assaying the lyophilocytes to identify the population or population Subgroup made within the group of lycaphocytes

L «Cercek and B, Cercek discuss in ia European Journal of Cancer, 3d. 17, 1981, 8. 167-171, ibid. Bd, 13, 1377, 3. 303-S-15 and la Biophysical'Journal, 3d. 23, 1973, pp. 395-405., The brightening and distension polarization jectra of -.- f'luorescein in living cells (the article of the Biophysical Journal) concerning the application of the pheoGien of changes in the Structured with the

-13- ·. ·

cytoplasmic matrix (SCM) in the diagnosis of malignant diseases. In short; The authors mentioned perform the so-called 3CM test after first trying to separate a specific lymphocyte subgroup from other lymphocytes as well as from other cell types by the density gradient technique.

This technique is - as stated above very unsatisfactory. First of all, it is very time-consuming, as judged by experts, and clearly evident from the cited papers by Cercek and Cercek, and secondly, as Cercek and Cercek note, the finally separated cells not only belong to the subgroup of interest on they rather include still, a large Ansah - up to 50% - of other lymphocytes _o Thus is: the analysis of their response to stimulation of the separated cells is very limited aussägefähige Third, and most important Finally, to obtain all of Cercek and Cercek performed excitation and reaction measurements on all the cells in a batch and not on a cell-by-cell basis, but it is clear that a cell-by-cell analysis provides much more information for understanding the biological Implications of the investigated phenomenon offers.

The present invention makes it possible to realize such analyzes very quickly and accurately. In this particular case, both speeds than .Genauigkeit also are of extreme importance, seven, one -enn the potential number of cancer Diagnoseuatersacaungen considered that should 'be carried out in each. Equally meaningful is that the novel invention, both systemically and methodically, offers possibilities of separating biological cells in which each

separated cell is stored at a known "address, to which one can then" - for the purpose of repeated cell observation and / or repeated stimulation sit subsequent analysis 'v / end can' ~ -.

liurz. In the present invention, a large number of cells, for example lymphocytes, in the blood, which can be assumed to represent one cell group or cell population ^s , are first of all other cells, i. separated from different cell groups or cell populations. In the course of the separation process the secreted lynaphocytes, in addition to being separated, are also separated from the other cells, each arriving at a known location * - hereinafter also referred to as the address "/ All separated lymphosomes are then selected on the same side Tests thereafter, each cell is separately examined whether, as a result of the test or as a result of a stimulation, it shows a special property, or if this is not the Pali * the address of each cell showing the said characteristic, is recorded "Thus, after 'aaesi' all the separated cells have been examined, the addresses of all those cells that have the special human senses are known. These cells represent a special subset of lymphocytes within the larger one.

T -trn '- * - \ W ^ τ "I"? "" ^ """* ^ 1B! 3" TI ^ · -T ¹ VUl ¹ ^ nO ~ s - <"*" ». * -" ^ / r - "S" * - V ώ! "ΐ- A ¹ ^""ί'Ό.""lÜV» T ^j : Ό "a V>*" ^J ^ nT>

j _tl j j ^ .ΐ 0x}, \ j ti · ν vj ^ jLi * "* - \ jr ^ u2 etu.i> y« ί '- ^ · * ·> u ΰ s WJW d- U , νί. - O ι ~ ιΚΖ - * - - ^. ^ - jji ο Ci ^. w * - -j ^! - l, λ- '- ^ - ^

De have been identified, kc: me-i - they are subjected to deni rest of lymphocytes to one or more additional iests zusa-cien, vvas but are exploring the Sigsnschciftsn cells of this or this sus-ät ζ union ¹ Ie st as a result .3, this can only be limited to the parts in the subgroup. Each line in the subgroup is individually examined, inceoi the instruments'

vidueile address directed v; earth * So as soon as the cells of the

SIS subgroups have been identified as sin'd, they are subsequently examined while all other cells belonging to the same group, but not being part of the subgroup, are ignored insofar as they are not subject to further investigation the subgroup has been identified, only the cells belonging to it are explored, thereby limiting the interference to only the subgroup cells of interest. "Moreover, since the study is performed on a row-by-row basis, it is more precise Data can be accessed for increased diagnostic accuracy "" Other benefits of being able to identify cells in a subgroup and examining each one individually are discussed below "

As already explained above, in a first step the lyaiphocytes become different from those contained in the blood. Cells separated. The separation is carried out using a perforated cell carrier 1, as shown in Figure iA. , , _

The line carrier 1 may have different configurations of openings or holes 2, which may also differ in their arrangement. In FIG. 1A, the holes are subdivided into rows and columns along the axes Z ' and Y,. are arranged. In Pig. 13, the punches have larger openings on their tops than on their undersides. In the embodiment described herein, the punches are dimensioned to be suitable for receiving lymphocytes, it being understood that lyophil in two main sizes - about 7 / um and stwa .10 »♦ .15 / uni - ·

Presence. At the upper surface or side of the support 1, the apertures have a cross-section of approximately 10 / um. The apertures on the lower surface or side 1b have a dimension approximately equal to 0. The walls of the apertures may be continuous or in-line Gradations - as shown in Figure TG - converge towards the opening at the base side 1b of the cell carrier

Generally, the opening should be like this. be designed to make the cross-sectional dimension less than 'at the top' either on its underside or on a cross-section between the sides 1t, so that a desired cell entering the 'orifice' does not pass through it; rather is held in her. Furthermore, it is important to narrow the thickness of the support between: the sides, and '1b so that the size of the orifice is' related' to the size of the desired cells, so that upon entry of a desired cell in an opening, practically the cleaned cell will find room in the opening, thus preventing the line from being washed out during a '' / '-' step - described below ''.

The shape of the two holes 2 allows the skins to be made by applying suitable means such as a pressure differential between the top and bottom of the beam or through. Applying electrons is more likely to be effective in holding forces. In short: first, to separate out a particular cell group from other cell groups, and since the cells of each group have known sizes differing from group to group in typical fashion, a carrier 1 with such holes is chosen so that when applying the different sells , gruooen containing substance -, for example, blood - on. the carrier 1 in an effective way 'the aeiaten ^ if'not even

all of the holes of cells of the group of interest, one cell per hole, are occupied.

As already stated, the holes 2 in the carrier 1 are arranged uniformly over or in the carrier - z. In rows and columns - to allow a unique identification of the position of each hole 2, for example, by its X and Y coordinates in the plane of the carrier su. In the described embodiment, the holes are arranged in rows and columns which extend at right angles to each other and on. in this way form a matrix-like structure, -The number of holes is chosen according to the number of cells to be supported. For example, with 100 holes per row and column, there are a total of 10,000 holes per shot, of 10,000 cells in the ¹ PaIIe of the embodiment described here, each hole having its unique position with respect to the X and Y axes. As such, the carrier 1 may have a circular circumference, as can be seen in FIG. 6C. As also seen su su, the carrier has a plurality of ears -8 to align the carrier in a holder 40. This device has a pair of grooves 9 extending from a groove in the top in which the carrier is held A bore extends axially beyond said groove in retainer 40. Other alignment devices such as pins or specially designed brackets are also within the scope of the present invention.

The support 1 is made of any suitable material, such as metals such as copper, gold, nickel, silver or anaer-i, or also of plastics with electrically conductive parts extending between the holes 2, as shown in Pig is shown. This way you can

the electric potential ends at any aellenthalt; In order to create such an interaction with the electrical charge of the cell, by controlling the voltage across different holes, the cells therein may be both electrically tied to the carrier and released from it.

j ) ·

For the realization of the method, a few drops of the solution containing the lyphosphates, for example blood, are dripped onto the cell carrier. The liquid enters through the holes in the carrier. The cells, however, remain on the support. "Since the sizes of the holes 2 are chosen to accommodate only lymphocytes, they are found to occupy the holes. Each hole harbors only one 'cell, over' and other cells are washed off the support surface, such as cells that are large enough to fit into any of the 'holes' and / or surplus, a, ., the number of holes exceeding Z-e'llen in order to prevent that the cells come out -again aus.den holes h can be attached to the carrier then this "this may ^be, take place using different means by ^v ^ be.ispieiswei3e Covering the carrier with an adhasable, cloidilizable substance or by electrically charging the carrier, but also by applying electric and / or magnetic fields

The further "combined method" of the isolation of the said population and of the simultaneous population of the population as a carrier is described below in connection with the Pig, 11,

M2A'unä 123 discussed «. '.

Each carrier provided with its group of cells of interest - eg B, lymphocytes - is placed in a support holder of a flow chamber, such as holder 10 (Figure 2D)

so. to provide the necessary environment for the test and measurement cycles to be described below.

In a first embodiment illustrated in Figs. 2A, 2D, 3A and 33, a plurality of dies or cell carriers 1 are placed on the holder 10 (Figs. 2D and 3A). It is only an orientation of the carrier possible, so that the perforations of the carrier with respect to defined axes, such as on the X and Y axes (see Fig. IA) are aligned * The upper part of the flow-through performing representing holder 10th i3t removable on one. The middle part Tt defines a plurality of channels 12, each of which is connected at both ends to a tube of a plurality of tubes 13 for the purpose of feeding and discharging a desired solution. The central part 11 is fixed on its underside by a lower part 14 (Fig. 23) of the flow chamber. This lower part comprises a transparent wall 15, which is required when incident and transmitted light techniques are used for analysis of the cells on the support. , '. , '

Fig. 3A is a sectional view perpendicular to the direction of a channel 12 (the solution therefore flows "into the side"). As can be seen from this figure, "a flow director 1S ensures that the reading comes into contact with the cell carrier, 1. In Fig. 33, a side view of this arrangement is shown schematically.

On the. Holders 10 are the carriers 1 of several different individuals (patients) in a way along the canals.

, '-20-, ·.

stretching row pla-siert, while, a column of straps of the same person extends perpendicular to the channels. Each, channel 12, corresponds to a 'start' so that the number of tests to be run determines the number of channels 12 in the ο,

Jed / ede flowing through any of the channels. Solution, therefore, moisten all in the vehicles over that channel? sensitive cells, which belong to a different patient. The cell carriers 1 can be covered by a glass plate 17 ', in order, if necessary, to allow the use of a liquid for the optical scanning system,. -. .. "" '' -

In Pig, 2A is a seven channels 12 comprehensive Durch-. In such a case, cells from each patient will emerge. carried seven carriers .- a 'carrier per channel' - while along each channel carrier with cells

/

of such different types of patients, as illustrated in FIG. 2D. Such an arrangement allows the stimulation of cells of different patients using different stimuli via each of the channels in either equilateral or successive sequence, then the reaction of each To further characterize or analyze cells, other descriptive embodiments include "'moreover, a variety of channels, so in a multi-channel embodiment, the number of cell-carrying carriers of each patient is typically the same However, the number of channels will be apparent from the following description. The present invention is not limited to multi-channel arrangements _. nachiclaoi ais stimulated by certain stimuli and studied, purified and so'in their before stimulation

As a result, only one cell-containing carrier per patient can be used, if desired. The cells on it can be traveled one after the other and. be flushed after each challenge and analysis for the next stimulation and analysis steps.

Before describing other embodiments of flow chambers, it is considered desirable to describe details of a preferred method for individually analyzing the lines which are placed at defined locations on said carrier and called into the Referring again to the SCM test as described by X. and B. Cercek et al. 1, and 3. According to Cercek, there are at least two characteristic features of a lymphocyte subgroup that are suitable for SCIiI-Te st. The presence of the specific antigen causes a lymphocyte to enter an irritated state when in a quiescent state. When fluorescein molecules are introduced into the lymphocytes using a well-known phenomenon known as fluorochromaticity.

phase to the irritated phase in-critical changes in the polarization of the fluorescence of fluorescein in the mentioned. Lymphocytes Lymphezyten in which stimulation procedures may evoke such critical changes, differ in at least two / characteristic "^ i ^ οο-λ ^ .- ττ c '-i tt" - η r <o Y ^ * ^ η " 3 ο ν »λ y- T - " Ί ^ η ¹ ^ η τ λ; tori »> η α τι 1 ί γ * ^ Ί H πργ

specific density and in the fact that for these cells a relatively high (.: ontroil-) 7 / ert of the fluorine-acid polarization is only "around a very narrow band of the emission spectrum around 510 nm."

, , '' -22-

is respected, "· ·

This second property is beneficially utilized to select the appropriate lymphocytes among the said lymphocyte population. mark or identify and thus circumvent the need-their, physikälisc.heii separation. It is thus the lymphocyte group which exhibits this particular spectral behavior, at which all further travel effects are examined, while all other lyophoics are neglected by the system of meshing of the system *. In other words, according to the invention, the carrier is first used. Helping the size of the holes in the carrier to separate L-lymphocytes in a patient's bloodstream from other cell types "The holes" are essentially filled with lymphocytes - one cell per hole, smaller cells move through the holes, larger cells become washed off the carrier surface.

The lymphocytes are then rinsed on the support with Ft) A + PBS, which is converted to fluorescein within the cells by fluorochrome. The pluorescence polarization is then measured and recorded for each cell within a narrow band of the emission spectrum around 510 nm. Only those cells, each of which has a relatively high value ^: the F ¹ ion s polarization - as? If the address of each cell on the bearer hole array is known, then the address of each cell belonging to the subgroup is also known subcellular cells, all ' ^l - * - Π O r * H 1 "? d ^ ¹ UEL · * "A nriyiA ^ rjtif '' ivjari ^ O'i!"'Cici ^ n-CQ ^r *> Π c \ / - "\ c \ ^ V>" ^ ^ i ^ * Λ " - ^ rl "^"'T ^ ¹ ^ "P Π CULL O" wj 1 j. iCi ^ piivj, Kj.'v' ^ i. ^ * α. w Ui-L ^ *. * i-J_ ^ ~ i-> ^^ * * -ί- * '* CJ O - ^^ vva * ^. v ^ - * -ÜA ^ X / ^ Vo. «» «- i. ^ -' ¹ ^, W ^ ' - - 'i -J- ¹ ^ _ ***. & Ζ ^ ^τ · _ «XJ.

be made only on the cells of the subgroup, while all located on the carrier and not the

Subgroup lymphocyte cells can be ignored insofar as neither measurements nor observations are made on them. Narrowing down the subsequent measurements or observations to just the cells of the subset shortens the analysis, which is again of great importance. Furthermore, and because of the fact that the address of each individual cell is known, the response of each cell can be recorded on each stimulus. This provides unique information that they were previously unavailable because measurements and observations were made on cell lots or on splitter branches. But even with a single cell under a microscope, one could not subsequently stimulate it with another stimulant and observe the subsequent reaction of the cell, due to the fact that so far one cell is not in a fixed "arrangement" with each the known address of the one cell in order to repeatedly direct the measuring and / or observation instrumentation to the same address for the purpose of observing the same cell. - ...

With regard to the minimum ratio of the polarizations measured at two fluorescence emission wavelengths - namely 510 nm and 515 nm - a suitable criterion can be determined. As a first step, therefore, the cells of the critical lymphocyte-litter group are identified by testing said criterion for each single cell on the support. Upon transitioning to a rice state, the degree of polarization of the irritated members of said subgroup decreases for said emission wavelength from 510 nm to one. Value of about 0.14. This change of the polarization ion wheel is only for the identified cells of the

  '- -24-

investigated subgroup.

In the following, in connection with Pig "4, a system for carrying out these tests for each cell on the carrier will be described. The. cells on the carrier 1 are added to

Next, it is typically rinsed with a solution of phosphate buffered saline (PBS) and fluoroscein diacetate (FDA). The latter substance is converted to fluorescein on the basis of the phenomenon of fluorochromania within the lymphocyte cell.

.- converts. The fluorescein is then excited by irradiation at the wavelength of 470 nm, whereupon it emits its characteristic / emission spectrum. The determination of which of the lymphocyte cells on the support belongs to the subgroup of interest is made by stepwise probing each single cell on the support using the optical analyzer 20 shown in Fig. 4,

'This'be contentV. a zirconium lamp (or laser) 2T, which serves as the light source with peak values at 470.1 nm and.., 468 nm, thereby eliminating the need for the use of a ^1- excitation filter to 'filter' any light of interest Range - d, h. 510 nm and 515 nm - not applicable. The light is linearly polarized by a polarizer 22 at right angles to the plane of FIG. 4, after passing through a converging lens 21a. The linear polarization is represented by the small circles »The linearly polarized light beam impinges on a mirror 23, which insofar. , . acts as a beam splitter as it transmits light-from / U> 500 nm, and reflects light below that wavelength.

^1, this V / else the light from the light source ¹ 21 via a \ "lens 24 to the transmitter 1 is thrown back"

The fluorine essence emitted by each cell on the support is separately measured and recorded. "The fluorine essence from one cell passes through mirror 23 and lens 24a to a Glenn-Thonison polarizer 25. Basically, the polarizer 25 divides the fluorescence into two parts : polarized an parallel to the plane of the paper (indicated by the hatching in FIG _e 4) portion which extends in the original direction of incidence, and the other polarized perpendicular to the plane of the paper (indicated by the circles in Figure 4 is marked.) part, which is deflected perpendicular to the incidence direction of · Each of the polarized beams is split into two equal and perpendicular beams by a beam splitter 26, 27. Each of these four newly formed beams passes through an interference filter of 510 or 515 nra 28, 29, 28 'and 29'j respectively, their intensities v / ground simultaneously by four photocells with microwave multipliers 30 "'31.

These four intensities are worn-in.einem computer sy st em deai as in Fig, 1-6 shown stored, and the degree of polarization for each wavelength, ie ^ t = 510 mn and / y = 515 nm is calculated. The degree of polarization is defined "as"

P = (I)) - I χ) / (I U + 1 J, )

liaah calculation of P-. _Λ and Pr ₁ - is their ratio, ie Ρ, -, λ / Ρπγ,? Jeiches the control value, calculated in 3chtzeit. The address of each cell with respect to its XY coordinates is known and stored in susatacien ait at its control value, ITa is examined for physical cells.

, their control values have been determined and stored, it is very easy to determine those cells which have a control value of not less than 1 , 3 "Ea are those cells belonging to the subgroup of interest, once this determination is made, v / ground all subsequent ,,

, Measurements or observations of the cells' response to different gag agents are made exclusively to the cells of the subgroup, while all other cells are ignored,

.. For example, only the. Re-examined cells of the subgroup to see which, of them, a sufficiently large change in polarization degree widened. to be able to recognize them as active and thus capable sur identification of a particular antigen,

It should be self-evident that the optical analyzer 2Q 0? Ig? 4) for examining each individual cell has an optical resolution in the range of one-to-many diameters. Object ic. achievable »The carrier with the cells is shifted step by step under the microscope from one perforation to the next, adjacent perforation; - That is a precise working mechanical. Yerstellsvstem - as described in 'ig »1's - required,

In another embodiment of the opt-laugh analyst, the necessity of the step-by-step retraction of the line, trough, is circumvented by the use of a laser as the source of src-ss. This embodiment is shown schematically in FIG. A laser beam 131 of suitable wavelength passes through a controlled deflecting optical element such as a rotating mirror 130. The laser beam 131 '' points. a cross section 'substantially corresponding to the size of a cell 1: z , by means of the rotating mirror 130, the laser beam 131 scans the cells in the holes in succession, exciting each cell one at a time, at each one

At a certain point in time, only one cell is hit by the laser beam, which is why only this cell at that moment emitted pluorescence light. The optical analyzer 131X mounted on the other side of the support 1 'has a field of view which covers the entire surface of the support 1. The L ¹ Io-. The reception of an emission signal corresponds to the intensity of the signal correlated with the position of the scanning laser beam 131, and thus each received and analyzed light signal is correlated with the position of the respective cell from which it was emitted. As can be seen from Pig, 5, the excitation takes place from the large side of the holes 2 in the carrier 1. For reasons which will be discussed below, for optical analysis, on the other hand, it is preferable to prefer that emissive which leaves the hole through the narrow end.

Having thus explained preferred analytical systems according to the invention, it should be understood that analogous stars can be used to measure other parameters as well, focussing on each single cell on the support is possible. Examples of measurable parameters are illuminance , optical density, refractive index, electromagnetic properties, absorption and scattering. In addition, the scanning procedure is not limited to beams such as visible light rays, UT rays, IR rays, and electron optical systems, but may also be probing include physical Ί-ion contact on each cell «/ other examples of measurable or observable properties include nuclear magnetic resonance (IiLiE), pH Y / ert as well as the Sellmorphology and their variations depending on different heating conditions«,

1 τ -γό - - '/ -' - τ - j ^ ρ "- ryi. ^ A'sfonq r". ~ ^ .-- 'nan "i,!!]; ¹ *; a>ionf" .p ¹ ' Λ Vi π μ η era X? «A"r> H- ~ 'μ ^ Ηρ ^ ί! -M ο η-τ' nia-o 'V α ^Α a ο .ο τ ". ο -y- · a -ΐ Ά Arns> r \ S ~ - Q η ρ Ί P -. '· - £ "-

Likewise, measurements of cell temperature and / or measurements of temperature changes can be made and recorded. In summary, any single or any of several measurable or observable characteristics of a cell can be on a cell be made for cell basis. Since the address of each cell is known, one can return to the same cell for the purpose of performing measurements and / or observations. * All measurements and observations on each cell can be recorded to provide uniform information for each individual. Cell to Obtain »This information can be correlated to gain insights and diagnoses of how they were unattainable,

The following is an embodiment of the-invention for practical clinical Sinsatz based on the ig * 6A _{0, a} oC explained, which for a variety of ~ a modified holder 40 - cable carriers 1 illustrate _o The retainer 40 is formed overall wavy to to allow its troughs 41 to be submerged in the solutions flowing through the channels 12 at a higher level of IT.The cable carriers mounted on the base of the valleys 4-1 can be moistened to allow them to be humidified Therefore, in this embodiment, there is no need for a flow director assembly, as described above in connection with Figs. 2A, 3B, and 3C As has been described in connection with the Pig, 2A ₃ , FIG. 2D, the cell carriers oriented on the same trough 41 belong to different patients. Nevertheless, there is no danger whatsoever ' mixed .vvmp.hosyten stimulus effect, because there is no physical connection ^: to the carriers, _i .

Even if a cell were released from a carrier, the chansen would be flushed away, much larger than the probability of being deposited on another carrier. "In Pig, 6A, carriers are merely internal. to see a wave trough. Practically, however, in each wave trough, there is a " ¹ carrier" for each patient.

In 5 ¹ Ig, 6C, the carrier 1 is shown as being detachable from the holder 40. However, in order to define its hole arrangement in.X and Y direction, it includes the eyelets 8 to be arranged in the grooves 9.

Since the cell carriers 1 of the present embodiment are immersed in the solution flowing through each channel 12, the microscope of the cell scanning optical system is provided with a quark sleeve 42 (Fig. 6B) which is mounted on the lens barrel. The channels 12 and troughs 41 are dimensioned to provide the relative loading required to scan the entire surface of each carrier. Movements of lens and carriers allow.

As already indicated above, the channels are initially supplied with a PBS + 5 ¹ BA solution for the purpose of selecting the Zeil subgroup on the basis of the control value described above. This is done during the control cycle to identify the appropriate cells located on each carrier and belonging to the subgroup. Thereafter, to determine the response of the cells read out to different rice means, each channel is assigned a; For example, different rice agents are supplied with phytohaleoagglutinin (PHA), SF, GaBP, tumor extracts, or any other desired ligand or antigen. Sedann studies and records the responses of only the cells that have been read.

In view of the above-mentioned irritants, it has been found that stimulation of cells by a stimulator does not affect subsequent irritation unless the stimulator has been flushed and / or neutralized prior to the next rice test, thereby eliminating any direct interaction or eccereboration In addition, it has been found that binding the cells to the carrier has no effect on their activation. As a result, the "irritation" procedures can be repeated on the same cell at the same site on the support, using different activating agents. "Thus, an ezactic profile of the response of each individual can be repeated. Thus, it is now possible to know the exact number and response of the activated cells and their locations on the matrix, this number being determined during and not 'changed' according to the measuring cycles described above.

Most of the Carrier Holders systems described above were designed to be scanned up, d, h, for "analyzing the S'luorescence light emitted from holes located on the uppermost side of the Carrier, thus allowing the use of the same optical system for optical inspection and analysis ^; in alternative embodiments, described below ^, the optical analyzer is arranged to read the passage of light passing through the narrow underside of the holes 2

receives'. In this way disturbing effects can be eliminated which are caused by the fluorescent emission of fluorescein, which in turn leaks out of the cells and is present in the surroundings of the cells. The light emitted by the surrounding fluorescein constitutes a ·. unwanted optical background. Viewing the '

Cells from the blistered sides or undersides of the holes allow a considerable reduction in this background, as the constricting cone acts as a shield against unwanted. Etnissionlicht acts. In addition, in the case of the entry of excitation light through the large openings or upper side of the holes, reflections may occur on the conical walls, which causes reflected light to strike the light as well as S ¹ , "a further advantageous effect of performing the optical analysis in the manner mentioned is that at each location on the carrier only the emission light of the cell enclosed in the corresponding hole is received, whereas other cells, which in exceptional cases may be present on the top of the carrier, do not influence the measurement results Another advantage derives from the fact that, because of the smaller orifice size in.praxi, it is much easier to analyze the smelter light from each cell separately without the risk of superimposition by other neighboring cells, ie the optical cell Systems over the holes not exactly working with extreme precision «

In the pig. _(7A, 73, 8 and 9 are shown various finishes: which make it possible to carry out the optical analysis in · the manner · explained above in a:... The first Ausfahrung for use with an optical analyzer in the form of a microscope (Fig 7A and 73), the bottom wall 110 of the bureau flow chamber comprises elastic glass holders (made of rubber) 111, each of the rialters carrying a glass plate -112 which is set on a cable carrier 1 disposed above the elastic glass holders 111 provide a liquid tight seal between the glass plate 112 and the bottom wall 110 of the passage chamber and

_i :, -32-

It is possible for the objective 113 of a microscope to be moved close enough to the cell carrier 1, around its individual one

'To be able to scan locations or holes from below (Pig. 7B). The lens 113 of the microscope is in its lower position '\, then the channel is on the DLirchflußkamnier

In a modification (Pig.8) of this embodiment, the lower part and side walls of the flow chamber are made in one piece from rubber,

In a second embodiment (FIG. 9), the optical analysis is performed from the upper side. The holder 114, for AÜe cable carrier A is, however, vice versa on a lower part .11 5 of. ' Furrow quiver-placed after being supplied with cells in a special device (described in connection with Figs. 12A and 12B), so that the conical holes in the carriers 1 are downwardly awakened .

holding the cells in place and effectively binding them to the carrier, a pressure differential is applied between the base "11, 5 (Fig. 9) and an upper part H o of the flowmeters, the fluid being in the 'Under part 115 has a slightly higher pressure 'than in the upper part 11 ο' 'Sealing strips ..11 7 protect the two parts of the' flowchainers from leaking '. When using this version, the optiache analyzer of Fig. 4'2 can be scanned the cells can be used on the 'supports 1', without omitting the advantages described above,

"'Let us now return to the problem of supplying the cell carriers' with cells of a particular desired population or group, which in principle is largely conventional

'How could be solved as described above. Figures 6, 6A and 7B illustrate a system-> simultaneous separation of said cell population

from other cell groups, which differs from the conventional unfavorable methods of cell separation.

The present embodiment also illustrates with respect to the separation of lymphocytes from the other blood cells for use in the SCM assays described above.

As in Fig. 10 and Pig. 11 can be seen resting in the .Furchflufikammer of Pig. 6a insertable holder 50 on an array of tubes 51 _}, each of which is divided longitudinally into an upper part 53 and a lower part 52. The lower part 52 thereby forms a low-pressure (air or liquid) extract to draw off liquids and inappropriate blood cells from the upper part. The upper part comprises bridges 54, under which drainage holes 55 exist and between which suction openings. 5o, which are aligned with the carriers 1 on the holder 50. In Pig "10" carriers for imaging cells of only one patient are shown. The upper and lower parts 53 and 52 are supplied with a liquid, such as a PBS solution As can be seen from the sectional illustration in Pig, 11, the liquid in the upper part passes under the bridges 54 and passes through them. By means of projections 5S, the liquid in the lower part 52 is forced to flow in the region of the drainage holes 55 and the suction openings 5c at a higher velocity, thus causing a local underpressure in these holes is dia.anfangs by the 'upper part 53 flowing liquid partially pulled through the aforementioned holes sum lower part,

The supply element 60 removably mounted on the holder 50 serves to supply the blood to the carriers 1. Feet Si attached to the insulator βθ prevents it from overriding

the liquid from one row of the slots 57 in the cold 50, to another row,>

Al 3 theoretical basis for. the understanding of cell separation using the above device is underlined by the following facts:

a) the size of the lymphocytes reacting is / 7 / u. ν ·. _: b) the size of macrophages, granulocytes is / v 20 / U, ♦,

c) the size of the "brothers" can reach 3 *. "5'U; '

of. exist large lymphocytes -. Ϊ 5 / Ui

e) the size of the platelets - negligible; ·

f) Cells can burst when distilled in

Water 'are left.

g) the lifetime of an erythrocyte in distilled T / aaser is much, shorter, than that of a Lvniphozvten,

The .Seilträgerhalt he 50 v / ill be first placed on the Höhrenanordnung, so that the carriers of the first row '(rechtv-inklig to the channels) ÜBS' ⁰ ran to 'the punches konuuec "· The encryption sorgungsvorrichtung oO is placed so, that their feet 6T are located on both sides of the cell carriers, the said -Sv.stem we zusuppigaetzetzt in the manner shown in Fig. 11. A syringe containing whole blood from a patient is placed in an S-2 opritsenhalter «In. Ig. 11, two such snrits are presented for two different patients «

The blood stream in each of the tubes is controlled by applying a suitable pressure to the syringe. * The first few pulses of pressure arrive at all the outflows of tubes PI and P2 (from two different patients). "

At a certain stage another pressure impulse will act on each of the. Rope carrier drops a drop of blood "The greetings of the holes in the support will not allow the blood to pass from one side of the cell support to the other, STunmehr in the lower half of the separation tube a negative pressure is formed by p33 - as described above these T-eil ces tube run IAST ₄ 'the blood -is ¹ ..gesogen immediately under the carrier. The smaller cells will pass through the support and suit the PBS flow. be washed away * Those cells that are similar in size to the size of the upper holes - al.so for example, 7 / um measure - _f v / ground remain on the support aad the largest are stored on the carrier "Ua locking of the carrier to Avoiding, the blood supply is quilted and PB's flows over the top of the matrix to wash off the larger cells. Most of them are sucked into the drawers. "The lower part of the cells passes to the next support (in the direction of flow) and will be discharged." Via, already explained, will be - '' ¹ U ^ "of I '" in "-" le ". U ^ sle ^ th ^ a- roping" he filled with the blood sines Soenders Llithin not there is the problem of Yerniischens blood unterachieölicher 3pender _o

In a next stage, the upper stream is stopped and another drop of blood dropped, on the cycle so often

The blood is subjected to a so-called "upper Pi.atswäsche".

The process is continued until the carrier in sufficient - • 'Maße'gefüllt is _o · This can be roughly checked by' be that the electrical resistivity of the carrier after each ^drop-1 is measured. Then distilled water can be passed through desired time point, which causes a bursting of the erythrocytes. The distilled water causes the ropes to swell, which is why the erythrocytes burst, while the lyaiphocytes solidify their seat in the carrier holes. At the "end of the desired set Seitintervalles PBS is introduced The erythrocyte released from the ruptured substances can not influence the Lyffi.ph.ozyten because ^r sih resistant Lb's'ungs st rom these substances;. Washes away" A -Electric loading or unloading of Matrix'oder the .Anlegen 'or. off of electromagnetic fields is also suitable How-the Vibieren the matrix by application of ultrasonic or other ¹ techniques that are equally genut.zt .könneia _e This procedure, in addition to the washing'. progressively of each patient P., P, etc., less than 1 cm.

This separation process takes at most about 5 sin. There is no restriction on the number of blood samples from which cells can be separated at the same time. Holder 50 is then ejected from the separation system. and in which the flow cocurrent shown in Pig. 4 is inserted, in which the operation of the optical ablation took place in the manner described above

A similar separation system which _, however, has been adapted for the holder 114 shown in Fig. 9, will be explained with reference to Figs. 12A and 12B. A base plate; Ver 120 is provided with channels 121 for fluid flow AEEN ~

see, which causes the required local negative pressure in the area under the supports 1 and the drainage oilers '55. For this purpose, 121 Voraprünge have been formed 122 on the bottom of the channels _β The holder 114 is such on the base plate 120, removably fitted that its carriers 1 are aligned with the projections 122 - seen in Figure 3 Ig "123 - that open their conical holes upwards, d.h. towards an overlying removable receptacle device which operates in a manner similar to that shown in FIG. 10. The supply device 123 is capable of supplying cells to cells by pressure only, as described in connection with FIG 10, however, it has been possible to increase the efficiency of blood supply by means of incineration devices, which, as shown in FIGS. 13A, 13B, 13C, and 14, are opposite to those of FIGS {Straps displaceable .sind. Turning now to FIGS. 1-3, 133, and 13G, an extension is shown with slide plates 124 extending across the supply device 123 * aligned with the channels 121. FIG.

At each outlet opening of a supply line, an elastic Verstreichelement 125 is arranged, as shown in the Hg » 133 and 130 can be seen. In 5'ig, 133 is a cross. ' The width of the resilient spreading device 125 essentially corresponds to the "side length" of a carrier and forms a small Ausgang.für the linear sweeping movement over the support surface, when moving the slide plate. 124 ₃ so that each support covered by a thin layer of cells 1st "Subsequently, the Waschschri-tte described above are superiors

nomraen "

FIG. 14 shows another embodiment of the spreading device in a cross section running at right angles to a channel 121. A blood line 127 is formed in a pivoting bar 126 extending along each channel 121, ^x which is attached to each carrier. , ner outlet opening is provided, which in turn has a distributor brush. When delivering blood to the carrier, the pivot bar 126 is pivoted several times, "whereby the cells are brushed onto the carrier 1, ^f .

While in the. In the above embodiment, the blood supply and the "G-rob" separation by means of a special Seoarierungs- device / is made, and then the holder 40, 50 and -optic sampling must be placed on a 'Durch, ui nnierier, it can s In some cases, as shown in Figs. 15A and 153, cell separation and the 'working' step are the most desirable. Optical scanning is therefore combined in an apparatus. '' ''. '

'Sin supply s sy stern 70 is superficially transversal zuein-

extending channels 71 _} 72 as well as also transversely extending each other inner lines 73> 74 verse- '' hen hen, at each junction is sin carrier 1 on a rotatable holder. Arranged 75 5. ein'entsprechender cross section is shown in Fig * 15B, A11 its'"base is a ^'sprocket; 7ö." Au3gebildet 5 which -'"77 cooperates with a corresponding rack, which in turn via the supply system 70. extends »Sine rack .77't rubs"

all brackets 75 of the corresponding column. The linear movement of this rack 77 causes the rotational movement of the brackets 75 * The direction of introduction of blood for coarse separation, -d _e h. for separating the group of lymphocytes from the other group of the blood cells, runs perpendicularly to the plane in which the cell carrier is scanned under the microscope. Thus, after the 'separation of the lymphocytes from other blood cells', the holders 75 for the Scanning operation rotated by 90 ° «.

In the above embodiment, to make the "diichi" technique possible (measurement of the light emerging from the bottom of a hole), it is, in part, a part of the channel containing the support 75 Crossing the cell carriers to form a longitudinally divided glass tube 73, this tube is arranged such that its open side faces the carrier (see FIG. 15B). In turn, a horizontal flow of liquid through the holder is made possible while transmitting light in a vertical direction. The negative pressure in this system is caused by the fact that the inner conduits 73, 74 are closed and thinner, while the upper conduits 71, 72 are wider and open. The same effect.can be caused by anderige.ige-. Techniques are achieved, such as by increasing the flow rate in the inner lines compared to the Durchflufemenge the upper. Channels <,

The procedure of FIG. 3 can be summarized as follows: The position of the supports 75 is determined by means of a 0, 90 ^W control device. In a first stage, when the coarse separation is performed, the channels 71 and conduits 73 are in operation, and at the end of this stage, the brackets are rotated 90 degrees. In this way, the channels 71 and lines 73 are blocked or -geisen

, "40-

closed, and the channels 72 and lines 74 are opened, ·

In this embodiment, a blood drip head may be attached to the scanning head - ζ, b. On the microscope. Then, in response to command signals from a controller such as a computer, the separation and the optical scanning are automatically performed, requiring no skilled operator. The operator only needs to insert the syringes - see Fig. 6 - as well as. Close the tests to replace the brackets 75 «·

In the Fig. 16, particular attention is drawn to an overall system of cell separation, sampling and analysis (diagnosis). Sine flow chamber 81 as described above is mounted on a table 82, which in turn is displaceable in three axes Σ>: Y, 2 by corresponding computer-controlled stepper motors 83, 84, .85. The optical system includes a microscope 86 having an optical analyzer 87 as described in Pig. For example, a sirconium lamp uses the same optical system in the reverse direction. A solution tank 89 stores all the solutions needed for cell separation and verification. The supply of the jewel time solution is monitored by a solvent control device 90 "To the Fluoressein-iConzentration in the cells welche'die affect absolute Bolarisationswerte -can stabilize ₅ below, an electro-optically ^ mechanical RUckkoppllängsregelung is used, in which the intensity of Fluorescence Soi3sional te3 periodically measured against a reference value Y / ird * Any deviation of the measured value from the reference value can be used to alter the FDA's consensus in the PBS solution caused ». '. ·

The analysis of the measured value can be made by any common computer system. Sine computer pre-stage 91 is used to convert measured values into computer-readable - usually digital - information. In a computer 92, the necessary calculations and recognition steps are performed and stored in a memory 93. A controller 94 downstream of the computer generates the control signals for the stepper motors and the solutions Control device 90,

The operation of the above system one can be summarized as follows: After the C-rob separation process, the flow chamber is mounted on the table 82. The microscope is stopped. From there. on, the test runs automatically »A PBS + F'DA solution is initiated through the channels and lines. Sin part of it pervades the carriers from the upper channels on the lower leads, and another part of it "continues its flow through the upper channels, washing off the protruding cells, ITach a chosen break 2 * B ₄ 20 min - the scanning begins «, the polarization of each

pin ^ pi ran ^p 7, p. ~] 1 λ? ΐή ν «/ 3 V; oi ri ^ n c'c -""H r, c η h ~ T, - π -7 ^ 1"! prsl äpr ^ i ^ isr.ac »con Pt" d 'la-pphi ¹ , - ³ S -.-! y ^T V λ »iac r \ -, c; i -h ΐ · - η asm. ? -] 1 _a C ¹ P Λ ^ Ό "ίΉρ r> r] = -" i

exciting light - ζ. 3. 470 mn - does not exist because the scan is done very fast- _a .

The optical information is input to the computer after conversion into an electrical pulse, evaluated and stored in the memory. In memory, each single cell is sorted according to their coordinates / d. h, the address

-ie r s: e s enriches β sells. <ef rs./τι werdaii ·,.

Collecting. Data can be summarized as follows: First, the control values of the cells are collected, all patients whose carriers are aligned in one channel, and then the scanhead is moved to the next channel (by lowering and moving the table sideways ) and used to determine the control values of all cells of those carriers aligned in the second channel. "Simultaneously with the data collection from the second 'channel, a rice solution is introduced into the first channel Channel is the sense head to the third channel far ₃ and a second "Re lslsüng is inserted into the second channel ~ ^! directs USWa. , · -.

Uach: the data collection from all carriers, the scanning head is returned to its first position. Then, the scanning operation on the excited cells is repeated. Now, it is a selective data collection whereby only those cells which correspond to the described optical criterion are scanned again. of the special subgroup ^ lithin-is the information collected in computers to determine the cell position, control values, polarization values after stimulation with PEA, post-irritation polarization values with CaBP, SCM- reaction ratio

1S7-171, 1981) ₅ polarization value after irritation with specific tumor stimulators and the like, *

The distinction between the cell carriers of different

folgerij as being the Grooseparierungsstufe on the

Brackets can be attached »A magnetic or optical reading mechanism can be attached to the optical scanning head, which can read the patient's code and input to the computer. All information relating to the respective patient can be stored in a pre-determined place in the memory of the computer.

This system can be used to determine the exact number of activated lymphocytes for each stimulant. The present invention provides a person of ordinary skill in the art of diagnosing cancer at a very early stage . Although the present invention has been described primarily in the Z iis aminen häng with the Kreb's diagnosis, it is still offens i cät Ii cn that the method according to the invention or the system according to the invention is not only limited thereto. In general, the invention imparts a hode and auxiliary -21Γ rapid implementation of biological examinations, again .which to novel clinical diagnostic and nimbly! ungsmo'glichkeit lead en ^v as well as to new Anwendungsmcglichke it en in the field of biotechnology -and biomanipulation.

As mentioned above, the exact position of each activated cell on a carrier is determined and subtracted,

σ η yes ΐ i-> h ρ μ- "..V · * '4- H -' η -ί a -f- pa γτ ^" Ί ί ΛΠ lins ~ a ·, · ' ^: ! π ς ^ ^ r 3 Π π ι -. ~ * π λ r \ Aor>

(j ϋ ύ "^ ^ iZIi-OC V ¹ J ^ -.. CIU i u-ö-ii iiijii <- · ώ -L j -... i ^ i- -> - ** w iJ Lj c: - jo-l w -!. »- ^ i OG-1 ρ '' - ji -σας i": rif-'oyrjr eil " ¹ ay. O r - ~ =, nn n Z -">"1?'1 V ^ ¹⁷ I -T ¹ ·· ->£;' -: - · = r · 211" i ^C 5 · '^ "' ^ ⁼ -

so that only the subgroup on the. Carrier remains, or by releasing and discharging only the belonging · to Untergrupoe.zellen cells from the carrier. For this purpose, one can make use of the confessed fact that ropes are not electrically neutral, but rather possess electrical surface charges. This fact can be summarized in the above-described embodiments or otherwise.

 '.. -44-

The same effect can be used in the selective release or retention of desired cells. This can be achieved by a modified embodiment of the cell carrier according to FIG. 1A, which will now be described in connection with FIG. 17 is explained. The outer shape of the carrier 100 corresponds to the shape shown in FIG. 1A. Carrier 100, however tnitelektrischen conductors 101 is provided extending between the holes 102 in a grid-like configuration and-the 'are electrically insulated "On the periphery of the carrier the conductors, in. A known manner (IC-technique) zi \ a computer-controlled circuit for selectively influencing the electrical potential of each individual conductor 101. In order to hold the cells in place, all conductors 101 can be held at the voltage which is exactly opposite that of the cell's charge potential , - which results in an electrical attraction of the cells .. For releasing any cell, for example the im'Loch a ¹ in Fig 17 _e located cell, the adjacent conductor ^ ₂ ^'Y x3' may ^V s1 "" ^Y y2 ^a suitable voltage ... be provided to cause the ejection of the cell as hole A of the carrier.

'. The Zaiiträger 100 can be applied using a multi-layer

  '. , -

ksnnt; "If an ionic solution or a PB3- is used, measures should be taken to prevent unwanted infiltration of potential surface charges on the support. Purpose, the holder can be coated insulating as well as ending in Sanal conductive elements.

-45-. ..

will see. This would cause an attraction and evolution of ions, which would prevent the formation of an ionic layer over the support surface, which in turn could affect the potential of the support. Another possibility 'is the use of nonionic organic solutions such as Verv / ending of lipids sura bathe the carrier in this Verfahrensstadiuin _o

The separation according to the invention of certain cells from all other cells is uniformly applicable in the field of clinical treatment in the production of clones, - clones can be made from certain cells which have been read according to the invention under ayadal ropes on the basis of any selected property. For example, it is well-known that the coroner of a person suffering from certain diseases, such as a person suffering from cancer, generates identifiable ropes which fight or kill the disease in order to be successful, but a great deal must be done. Such - hereinafter referred to as killer cells - ropes in the body be present. With the present invention know blood, Lymphknoten.und diverse Korpergeweb.e which some Kilierzellen

I ¹ Ts c rid -sol, as in the above-mentioned reference, "- of which alone; -"; -I c, ^ c ^ ^ f ¹ II ⁰ H. ^ PP ^ ^ "i 3" ¹ ^ -Y ^ r »^ aj2. 3 in ^ '' CO 'Ι-Γ' ¹ '-M Θ. H i ei. ^^^ ϊ ^ ^ j ^ rigjj Kiiierseiien comparable by suitable Zelivermecrungstechniken

the original selenium was also taken from the disease. In such a jelly there is no z-elliptical

ha '"S ¹ C ^ AM i Π" ^ H "ΐ ^ OA *" o 1 Q 3! renn Π "ϊ λ ^Τ / Ο"' ^Λ ^ "* ^ ¹ ^ ^G " 0_ ° ^ j ^τ "T * 1 .Π ΓΪ? '1 ^

to be used to supply the body of a L.enseher: -with sufficient cells to ooze the disease of this very body, \

It should be noted that, in addition to the previously discussed 'separation of interest and other ropes, either by ejecting or removing the cells of interest from the support and leaving the remaining cells alone on the support, but by eliminating them the non-interest cells and the 'remaining, only the cells of interest on the support also the possibility' is to realize such a separation if desired, that the non-interesting cells are destroyed as by k k while -i they-in the öffnungen- the carrier bef domestic 'so that only the interesting ropes alive'

remain on the support, 'killing the in the openings r \ α τ

e fin dl ic. hen ropes, _d's h #-in-situ, can by directing a laser beam to d "'s ieweili ~ e, with their address loading line knew done," but there may be similar or analogous ways apply * one killed, - ά, higher-dead ropes can -somit -. ^ f you own is-yet on the carrier - are considered to be separated or eliminated.-as it once it is killed, fails for all practical purposes * The term "ejection" of a rope is thus understood in the present context in the sense of removing a rope from the wearer or killing in-aitu * -

i7as the Multiolisieren interest ropes _concerns} 30 should be clear that this can be done-j after:

(a) remove the repetitive lines of carriers carrying dec live but not interested lines.

(b) remove the non-interested ropes from: wearers and thereafter "the ropes of interest" auni Swe'cke.

c) the non-interest cells still present in the openings of the carrier have been killed, whereupon the cells of interest are multiplied in-situ, ie during their presence in the openings of the carrier _o

Although the principles of the present invention have been described with reference to specific systems, forms of application, and methods, it is to be understood that this description is intended for purposes of illustration only and is not to be construed as limiting the scope of the invention ,

The present invention is expected to yield numerous new fields of application in biological research, clinical treatment and industrial production, and has already been shown to a satisfactory degree by optical parameters which are the cyclic phase of the cells to be occupied by the present invention, it is possible to have a cell population in terms of the ZsilalterSj the cyclic stage and him _; A clinical application possibility of the above is the possibility of early detection of the leukemia, by the presence of a large number of young ropes of a certain type bs, "". of certain types in the blood and ICnochenmark.

As a further clinical application, immunoreactivity assays for organ transplantation can be performed. For this purpose, a preparation of the graft is used as an irritant in the method according to the invention. .

A general and priority feature and advantage identifier. It is a sign of the invention that the time required for biological experiments and tests is significantly reduced since cell identification and testing is performed in a much shorter time than is the case with conventional biological methods in which ^v natural developments often have to be reproduced under artificial conditions, resulting zu'ungenauen results, which in turn make elaborate statistical analysis necessary ^ the invention reduces the "!! infill se of a numerical analysis-permitting conditions with a minimum of statistical to v / and" Compared with the prior art, the need to perform measurements in absolute margins is very short, using the method of the invention, which in turn reduces the effect of changes in ambient conditions such as temperature, humidity us ^ v, reduced «'^;' ^

Although specific embodiments of the invention have been described and illustrated herein, it is to be understood that the skilled artisan readily devotes his attention to variations and the variations thereof

Claims (8)

.-49- Srfindungsanspruch 1 Apparatus for selecting specific biological cells from other cells and observing at least one selected property of the selected biological cells, characterized by an arrangement of support means having a predetermined distance between an upper surface that is loadable with the biological cells and an opposite bottom surface , comprising a plurality of recesses extending in predetermined order between both surfaces, the recesses having a predetermined configuration with predetermined dimensions at the top and bottom surfaces ^s , which are respectively definable as top and bottom dimensions, and when loaded of the upper surface with biological cells only selected cells of predetermined dimensions are kept substantially in the recesses with essentially one rope per hole. " 2. Apparatus for observing at least one selected property of selected biological cells according to item 1, characterized in that, in the case of the support means, the bottom dimension of each recess is greater than the smallest dimension of the cross-section. "3 V ^ T ¹ Τ- ^. Γ · '' - ^ - ^ V] ^ C H α t ~ * *" ΐ "" H ^r - £ "?. ir * v - ^ ο γ * O st- ^ '£ ο' f \ ^ T "p, S ^ q ^ jJTI3 'ij ία.S, d U. 2 OJP..? that upper and lower body measurements of each recess in the Liikro- ^; met erbereich read: _e  Ό " 4 »Device according to item 1, characterized. in that the thickness of the support means and the upper dimensions of the recesses are on the order of the "^ ¹ ^ 'selected cells. 5 »Device after. Item 4, characterized in that the recesses are dimensioned so that they can accommodate lyophocytes, namely a pro-recess, 6, device after. Point -1, characterized in that the arrangement further comprises first structural elements for supporting the support means at a certain location thereof, each recess of the support being in a known location on the structural elements, is definier'oar 'as the' best place ', - instruments for observation and / or measurement of at least one line. ^: property as well as st the instruments ¹ j to .the instruments to any .Each recess bezogen.auf their specific place, * set su can and to be able to observe at least one property of the cell therein -or- measure *; '·. Apparatus according to item 6, characterized in that the instruments include optical scanning instruments for determining optical "characteristics of any of the cells". 8. Vorrichtu-ig according to item-6 _}, characterized in that su. The first Sxrukturelementen means for mounting ainpyi 7 "Ϊ al r nl ·, -, -. A ""· ο nrp"] τ ρ Vi ττλ i "^ - 'η α π η ir ¹ +"^; aTKail savings ¹ -aufleisen, as well as second 'structural sieves present sixrd, dis with regard to, on the first structural element so. that the cells ¹ on the spatially separated support means can be stimulated simultaneously by a given substance, so that the control means are able to move the instruments to the particular location of any one of the recesses -P ivm-on ^ ai: · "ο ν-ϊ iaw Φ γ> ο ο ο r> s ί .. η r · · : ι ο = Π" .Π ¹ f Vrh S Π. γ> ο ο · ο r> dv ^ ^ S η *** ο "τ ¹ ί ό; -31 ί λ '^ ^ -" 7. Ci 1 1 it ^ n 9, device according to point 3, characterized in that ... the first and second structural elements are mutually adjustable and one another, the cells being stimulable either from above or from the bottom of the recesses are by moistening them «. '. · '. 10 «Device according to item 8, characterized in that the second structural elements are those which contain different stimuli. and the first and second structural elements are mutually adjustable, wherein cells in at least some of the spatially separated support means are stimulated in the same direction by means of the different stimulation means.  11 β device according to item 1, characterized in that the carrier means include force-generating means that hold any of the cells to the 'recess in which they are arranged, or pushes them out of this « 12, device according to item 11, characterized in that the force-generating means include those which are located in predetermined profiles with respect to the recesses to connect any asr cells to the recess ^; in which it is arranged to hold by means of electromagnetic devices or to carry them out of the recess, 13, device according to item 11, characterized in that the force-generating means include those which produce a pressure difference across at least a selected part of the bearing means. 1.4 'device' to point -11, characterized by, that ¹ to. the force-generating means include those which increase the adhesiveness of the cells in the preparations. 15 * Device according to item 11, characterized in that to. the first structural elements include such sum supports of a plurality of spatially separated support means, each containing cells in their recesses, and 'second structural elements,' associated with the first. 3structure elements are selectively adjustable to allow the. Cells on the spatially separated support devices are stimulated simultaneously with predetermined substance, with controls that are in position, the instruments in the specific location -, any of the recesses or any of the carrier. to observe the "ropes in it.". '' '' - '. '' - 16 «device according to item 15», characterized in that the. first and second structural elements can be adjusted against one another or to one another, wherein the cells are either of; 'to be stimulated above or from the bottom of the savings by moistening.' ; 17 "" Apparatus according to item 15, characterized in that su    '. < the second structural entity with different, : 'which include stimulating substances in it, and the first: and second structural elements against each other or' are adjustable to each other, -wobei the cells in little ^; ·. at least some of the spatially separated carrier devices are stunted by the different stimulatory substances " A method for separating at least one specific group of biological cells to facilitate the observation of selected cells in the separated group, characterized in that the top of a support having a predetermined thickness between the top and the opposite bottom side is filled with cells wherein the carrier has recesses in predetermined order lying between said sides, and when the carrier is substantially completely covered by cells only selected cells based on their dimensions are held substantially within the recesses, where id is substantially only one cell per recess is given. ·. 19 · Method according to item 18, characterized in that; continue to sweep the top of the support to remove any cells not deposited in the recesses, essentially only the! separated cells remain in the carrier recesses, with essentially one cell per recess. .20. A method according to item 18, characterized in that further as a puncture of a known cell characteristic, any cell is held in or out of the recess in which it is deposited. ' 21, method according to item 18, characterized in that the. said support means is stored in predetermined orientation on a support structure ,. the location of each recess and the cell therein being definable, as the location of the cell is known to control the instruments to each of the mentioned. Each one of a number of cell properties can be observed and   , -54-; '... is determined, including those properties, the aas of their, stimulation by the predetermined substance results, animals, - / '' 22. Method according to item -21, characterized in that the cells _; on the carrier ^ after a previous stimulation • treated in such a way that they return to their previous state, 23, method according to item ..2Z ₁ characterized-by further comprising the steps of: neutralizing the Cells on the support after previous stimulation and at least stimulation of the cells by different substances. , ..24 · .-- method according to item 13, characterized in that further the cells which have been separated, propagates, be around. to obtain a higher number of separated cells »' _i ''·: 25. Method according to item 20 ,. characterized in that the cells that are propagated are the cells that. from , be removed from the carrier devices «'ν. 26. The method according to item 20, characterized in that the. Cells that are .repleted are severed cells, which are ^: remain on the support means. 27. Method according to item 26, characterized in that the cells are used while their positions are maintained in their respective recesses. 28. A method according to item 18, characterized in that furthermore at least one cell which is not among the separated cells is killed while the cell is in situ (in situ), namely in a recess of the carrier. '. , ': 29. The method nach.Punkt 28, characterized in that further the proliferation of living cells on the support , - · --- takes place on the spot (in situ). ' , "Method according to item 29", characterized in that the propagation process of at least one cell propagating in situ continues to be observed » For this 12.Seiten drawings