DE2153405A1 - Device for determining the percentage of particle types in a medium - Google Patents

Description

6806 TECHNICON INSTRUMENTS CORPORATION, Tarrytown, NY, USA,

Gate direction to determine the percentage of Types of particles in a medium

The invention relates to a gate direction and a A method of determining the percentage of a selected particle type within one in a medium contained group of particle types, in particular for determining the percentage of selected types of leukocytes in a blood sample, i.e. for precise leukocyte differentiation.

The exact knowledge of the proportions or populations of different Types of leukocytes in human blood form a cornerstone of diagnostics. The different Namely, leukocyte types have different particular punctures in the human body, and the Knowledge of the proportions of the various types of leukocytes in the blood often provides valuable information special disease processes, for example whether it is infection, inflammation, immune or neoplastic Diseases.

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There are three basic types in whole blood of cells, namely erythrocytes or red blood cells, leukocytes or white blood cells and your platelets or Platelets. Used to differentiate and count the different types of leukocytes in whole blood samples numerous processes are already known. In general, the differentiation and counting is done manually by being skillful Laboratory personnel made. It becomes discolored that has been dried on a cover slip or microscope slide Blood smear screened visually. Such methods are for example in'laboratory Medioine-Hematology ", third edition, J.B. Miale (The CV. Mosby Company, St. Louis, Ho., 1967). When staining, the cytoplasm surrounding the nucleus of the leukocytes and the nucleus itself is differentially colored so that spectral and morphological information can be obtained. In general, the leukocytes are divided into two groups, namely granulocytes and agranulocytes. The granulocytes can be identified by the fact that they contain granules in the cytoplasm which are opposite are selectively sensitive to special chemical dyes. The agranulocytes have a proportionate clear cytoplasm with comparatively few granules. The agranulocytes can therefore be stained by staining differentiate less well from one another. Han can, however, based on the morphological properties of the agranulocytes make a differentiation.

The known manual methods of differentiating and counting leukocytes in blood samples are extreme laborious and time-consuming. Generally, a small volume of blood sample is streaked on a clean slide. After the blood sample streak has dried

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it is treated with coloring chemicals and fixing agents, such as a Wright agent. The smear is then screened under a microscope, revealing the types of leukocytes brought into view individually differentiated and counted. This procedure is extremely tedious and human Prone to errors. Furthermore, only a small number of leukocytes have been screened, generally no more than a hundred. Since leukocyte differentiation is based on such a small number of leukocytes, Moreover, one only gets a rough approximation of the true percentage of the different types of leukocytes Recently, the visual method has been replaced by an electronic scanning method, where between the microscope's field of view and a Computer suitable scanning and measuring devices are arranged. This electronic process can be called pattern recognition process, in which to differentiate very many parameters of the individual leukocytes are measured, which are brought into the field of vision. Because of this parameter identifies and classifies the particular type of leukocyte by the computer. Regardless of the achievable Accuracy is one such electronic pattern recognition method for accurately differentiating the Leukocytes extremely slowly, as the colored blood sample has to be scanned according to a special pattern, which is either is adjusted manually or mechanically, often by the laboratory staff after a hay adjustment refocusing must be performed to accurately measure a particular leukocyte. The high focus range and the scanning of the slide can be done automatically, but this is extremely complex. Without this automation, the procedure no particular advantages, since the counting speed is due to the scanning and refocusing

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is low. With today's pattern recognition devices, a skilled laboratory assistant can manage around 10 cells per Determine the minute. It is therefore extremely time consuming and expensive to accurately determine the percentage of different types of leukocytes in a blood sample create long series of counts based on solid statistics.

It is also known for leukocyte differentiation to use an aqueous or non-aqueous sample of blood Suspend liquid and send it through an optical or electronic arrangement. However, if you can When staining processes are used in these arrangements, there is no selective discoloration of the individual leukocyte types achieved. With these known arrangements one can therefore only determine the total number of leukocytes and fail to properly differentiate or sub-classify. There is a differentiation only possible due to various morphological properties.

Except for the tedious and tedious manual process and except for the use of extremely complex and expensive equipment, these are the known methods and devices incapable of extremely accurate differentiation of the various types of leukocytes in a blood sample to determine the percentage populations on a healthy statistical basis or numbers of the individual types of leukocytes. A main reason for the disadvantages in the known methods and devices, is perhaps to be seen in the attempt to differentiate between the various

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Types of leukocytes in a blood sample with only one Make a group of parameters of the cell, namely only using the morphological or spectral information. Furthermore, the known methods for precise differentiation are therefore extreme it has become complicated that one tried to find out about the various Identify types of leukocytes in a single volume of blood sample simultaneously, using the observed individual cells one after the other. This will the possibility of errors in determining the individual proportions of the various types of leukocytes in a blood sample considerably increased

The invention is therefore based on the object of providing a method and a device for precise and simple Differentiation of the different types of leukocates in to create a blood sample.

The leukocyte differentiation should be carried out automatically and also extend to a subclassification of leukocytes.

The aim is to determine the populations of one or more types of leukocytes present in a blood sample carried out at the same time.

Furthermore, there should be the possibility that automatically numerous successive blood samples with respect to different Leukocyte types can be differentiated.

Both spectral and AUOH morphological information to differentiate and identify particular types of leukocytes in a blood sample to be used without human intervention ·

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Furthermore, there should be the possibility, on a selective basis, of the population of a particular type of leukocyte to be determined in a blood sample.

Furthermore, in the method and the device, one or more should automatically be present in a blood sample Types of leukocytes can be differentiated and their populations determined, on a selective basis and regardless of the volume or flow rate of the blood sample passed through one after the continuous Flow principle working arrangement flows.

In addition, the method and the device should be suitable for the proportion of one or more types of particles to be determined in a liquid medium.

According to a preferred embodiment of the invention, several blood samples are introduced successively in the form of a stream into an arrangement operating on the continuous flow principle. The basic structure of an arrangement operating according to the continuous flow principle is known, for example, from US Pat. No. 3,421-432. The supplied sample stream is divided into several partial streams. In each partial flow, the erythrocytes are hemolyzed and the leukocytes are fixed. For this purpose, suitable chemicals are continuously added to the individual substreams. Along with these chemicals are added coloring chemicals that are selective for a particular type or group of leukocytes. As a result, only one particular type of leukocyte or group of leukocytes is colored in each partial flow. The selective staining of a particular ax or group of leukocytes in each

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Partial flow allows the stained cells to be stained directly and extremely reliably by automatic means can be counted. This allows each type or group of leukocytes by a single one Identify parameters, so that the sampling of numerous parameters and the mutual relationship of the Parameters for identifying a particular type or group of leukocytes are omitted, as is the case with the known one Pattern recognition process of EaIl is.

According to a feature of the invention, which is also called volume detection, one in each of the substreams equal predetermined number of leukocytes, both colored and unstained, counted while at the same time within this predetermined total number of leukocytes, i.e. within a reference range, the colored ones Leukocytes are identified and their numbers are determined. Counting the total number of colored and unstained leukocytes and the counting of the number of stained leukocytes in the partial currents is carried out in parallel and made in phase. This allows you to see the results obtained in relation to the same blood sample correlate them with each other. The volume and the flow rate of the sample in each of the substreams are therefore insignificant and do not affect the percentage of stained leukocytes.

In each substream, a particular type or class of leukocytes is selectively stained so that the presence each stained leukocyte by conventional absorption techniques can be determined. Counting the stained leukocytes in the individual partial flows can can be carried out in parallel and at the same time. So that the individual partial flows are related to each other

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can put is the number of leukocytes, namely both the colored and the non-colored, determined simultaneously by conventional light scattering methods. You will be stray signals with regard to each particular Partial stream processed independently of each other · While the scatter signals are being counted, the Absorption signals counted to the number of stained leukocytes within the number of stained and not to obtain stained leukocytes. If a given

"Number of scatter signals has been counted no further selectively stained leukocytes were counted in the relevant substream. So the count will be completed. The absorption signals that detect the selectively colored leukocytes in the special partial flow, are therefore only counted within the time span required to count the specified number of colored and unstained leukocytes is required. The detected leukocytes flow through the field of vision a flow cell. The end of the counting process in the case of a particular partial flow, however, does not mean that the counting process also occurs in the case of the other partial flows is interrupted. Each partial flow is therefore assigned its own counting process. When the counts at all. If partial flows have ended, the ratio of the count values of the scatter signals and absorption signals provides the percentage or population of the particular type or group of leukocytes in the blood sample. the individual counting results can be correlated with one another and recorded. When a certain group of Leukocytes can be counted by means of suitable logic operations between the parallel counting channels or special types of leukocytes within the canal due to pulse height discrimination

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"Differentiate certain group. As a result of the volume verification is the arrangement against flow fluctuations nabezu insensitive in the individual partial flows. In addition, the size of the volume of the blood sample plays a role no role in the partial flows ..

According to a further feature of the invention, the scatter signal is used for multiple punctures. Since the scatter signal is the Presence of a leukocyte identified, namely either a colored or an unstained leukocyte flowing through the viewing volume of the flow cell, each scatter signal defines a sampling period and is therefore used as a control signal to control the channel logic actuate. This will provide a more reliable statistical basis for determining the population of the Leukocytes reached because you can use the scatter signal to sample the absorption signal, so that electrical Noise signals are not taken into account.

An automatic method and a corresponding device for differentiating and determining the populations of particular particle types in a medium, for example of leukocytes in a whole blood sample, are thus distinguished according to the invention in that a number of in-phase rapid currents, all of which have a surge containing the blood sample, are treated in order to stain either a certain type or a certain group of leukocytes, and that each partial flow is passed through a respective associated viewing chamber. In each batch of sample that flows through the assigned viewing chamber, the number of colored leukocytes and the number of colored and non- colored leukocytes are simultaneously counted by photometric devices. The ratio of these

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Counts give the population of the particular leukocyte or group of leukocytes in the batch of samples at. Preferably, the counting of stained and unstained leukocytes in the batches of samples is related to made a predetermined measure so that one can determine the populations of the particular leukocyte species or the particular Can directly relate groups of leukocytes to one another and record them in a correlated manner. As a result of the The results obtained from the flow rate or the volume of the sample batches are also based on the specified reference dimension completely independent in the various partial flows. Furthermore, one can, if special leukocytes one the same substream are colored and counted as a group, Determine the populations of the individual leukocytes in the leukocyte group using logical comparison operations «

A preferred embodiment of the invention is described with reference to figures.

1 shows how the Figures 1A, 1B, and must be assembled 10 to obtain a complete image of the embodiment.

Figure 1A shows a schematic diagram of a multi-channel arrangement working according to the continuous flow principle, in which the leukocytes of a whole blood sample are optionally stained.

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Figures 1B and 1C show in the form of block diagrams

Logic arrangements which occur in the arrangement according to FIG. 1A Count different types of leukocytes and the relevant percentage population of these leukocytes determine in the whole blood sample. The logic arrangement according to FIG. 1B is used for the Eosinophils, lymphocytes and neutrophils, while the logic arrangement according to FIG. 10 is intended for the basophils and monocytes.

As shown in the figures, the analyzing arrangement includes a sample supply device 1, which is sequentially supplies individual blood samples to a line 3. The sample feed device 1. shows a step-by-step further switchable turntable 5, which carries several sample containers 7 in a circular arrangement. For gradual Further switching of the turntable 5, a drive device, not shown, is provided, which works that the sample container 7 is brought one after the other to a sampling point under a suction probe 9 will. The suction probe 9 is controlled by a suitable mechanism 11 which is constructed in this way, for example may be, as is known from US Pat. No. 3,134,263. Thereafter, the suction probe 9 is immersed during the standstill of the turntable 5 into a sample container 7 located at the sampling point to a part aspirate the sample in the sample container and feed it to the line 3 for subsequent treatment and analysis. Lifting the turntable is a thing of the past

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container 13 arranged. The suction probe 9 is controlled by the mechanism 11 in such a way that it is immersed in the washing liquid container during the indexing of the turntable. The line 3 is connected to pump hoses 15, 17 and 19 of a peristaltic pump 21. The pump hoses are continuously pressed against a plate 25 by pump rollers, which are symbolically indicated at 23. The fuel supplied via line 3 power consists of successive blood samples * come from neighboring sample containers 7 and are separated from each other by an air thrust, a washing fluid thrust and a further air boost "Since the line 3 via a branch piece 27 to the pump tubes 15, 17 and 19 is connected, the supplied stream is divided into partial streams, which consist of a part of each batch of the original sample stream and are fed to various analysis channels for selective staining of the leukocytes »

According to the preferred embodiment of the supplied through the pump hose 15 part-stream is for dyeing \ and treated counting of neutrophils and eosinophils, as a class in Analysierkanal I and further for selective staining of eosinophils in Analysierkanal II · The partial flow in the pump tube 15 is initially made with a current mixed with a suitable chemical to fix the leukocytes. The fixative can be 10 # formalin in a 0.2 molar. Act sodium phosphate buffer with a pH of about 7.0. The fixing agent is sucked in from a supply of fixing agent (not shown) by a pump hose 29. The fixing agent supplied via the pump hose 29

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is sent through an ice bath 31, the temperature of which can be 4 degrees Celsius, for example for about 5 minutes. Air is sucked in via a pump hose 33, which divides the flow of fixative before it is fed to the sample partial flow and mixed with it in a mixing coil 35. The fixative is initially cooled in order to delay the chemical reaction with which the leukocytes are fixed until a complete mixing of the fixative flow and the sample partial flow has taken place in the mixing coil 35. The partial flow coming from the mixing coil 35 is heated in a heating coil 37, for example for about 3.5 minutes to 51 degrees Celsius, in order to support and accelerate the fixation of the leukocytes. The fixed partial flow coming from the heating coil 37 then flows through a cooling coil 39 »For example for about 2 minutes at a temperature of 4 degrees Celsius in order to prevent a further chemical reaction and to open up the possibility of introducing a suitable lysing agent, for example acetic acid 10 56 in HgO. The acetic acid is sucked in via a pump hose 41 from an acetic acid supply (not shown). Behind the cooling bath 39, the lysing agent flow is combined with the fixed partial flow. The resulting current is sent through a heating bath 43, for example for 1.5 minutes at 51 degrees Celsius. This is to render ineffective any catalase that could interfere with the subsequent chemical reactions, for example the preferred use of hydrogen peroxide. Furthermore, the lysis of the erythrocytes is to be ended, ie the hemolysis by the acetic acid present in the substream. The fixed and lysed partial flow coming from the heating bath 43 is divided again and over

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Pump hoses 45 and 47 are fed to the analysis channel AI and All, respectively. The partial flow supplied to the analysis channel AI via the pump hose 54 is initially mixed with a suitable substrate, which contains, for example, hydrogen peroxide and a solution of 4-chloro-1-naphthol, and with a solvent of 2,2'-dihydroxyethanol, buffered at a weak acidic pH of about 4 »5 _f, which is referred to in the following as dark colorant. These substances are sucked in from associated supply sources (not shown) via pump hoses 49 and 51, introduced into the partial flow and mixed with it in a mixing coil 53. In order to accelerate the mixing, the dark coloring agent supplied via the pump hose 51 is subdivided by an air stream supplied via a pump hose 55.

The air supplied via the pump hose 55 forms individual air bubbles in the dark dye flow, the frequency of which is significantly greater than that of the intermediate sample batches in the partial flow, so that the individual blood sample batches are further subdivided. This further subdivision is known per se and ensures complete mixing of the various liquids when they flow through the mixing coil. Furthermore, the additional air bubbles in the stream promote the additional cleaning of the inner walls of the lines in order to prevent contamination of the subsequent batches of samples.

The dark coloring agent supplied in the analysis channel AI via the pump hose 51 and then subdivided by the loft supplied via the pump hose 55 is

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with a flow of hydrogen peroxide (H ₂ O ₂ ) supplied via the pump hose 49, which forms the substrate. The substrate stream is thoroughly mixed in the mixing coil 53 and is reactive in order to optionally stain both eosinophils and neutrophils, as is described, for example, in a simultaneously filed patent application VStA., October 30, 1970, 85 333, Ansley et al. After the mixing in the mixing coil 53, the substrate flow is brought together with the partial flow supplied via the pump hose 45, which is fixed and lysed. Complete mixing takes place in a subsequently arranged mixing coil 57. The mixed flow then passes through a heating bath 59 , for example for about 1.5 minutes at 37 degrees Celsius, in order to accelerate the coloring and the color development of the eosinophils and heutrophils. This color development is described by the following chemical equation.

4-chloro-1-kaphtol + H ₂ 0 ₂ --i | £ 22i ||| 2- HgO + black heather

After the development of the ear, the refractive index becomes the adapted to the red cell walls, for example by adding propylene glycol via a pump hose 60 is supplied and mixed in a mixing coil 61 with the partial flow. The treated in this way Partial flow passes from the mixing coil 61 via a line 63 to a flow cell arrangement 65 to to count the colored leukocytes in the substream.

A similar treatment is carried out in the analysis channel All in order to selectively color the eosinophils in the partial flow supplied via the pump hose 47. The eosinophils are counted to, for one, the

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to determine the percentage population of eosinophils in the blood sample in question and, on the other hand, to determine the percentage population of neutrophils by subtracting from the group result obtained by counting the eosinophils and neutrophils in the analysis channel AI. In the analysis channel All, the partial flow supplied via the pump hose 47 is mixed with a substrate, for example consisting of hydrogen peroxide and a solution of 4-chloro-i-Napbtol together with a solvent of 2.2 ^I -dihydroxyethanol, buffered to an acidic pH- Value of about 3.5, called acid colorant in the following, and supplied via pump hoses 67 and 69, respectively. The acid colorant supplied via pump hose 69 is subdivided by air supplied via a pump hose. In a mixing coil 73, the streams of acid colorant and hydrogen peroxide are completely mixed to form the substrate. The substrate flow coming from the mixing coil 73 is combined with the partial flow supplied by the pump hose 47, and then the combined flows are mixed in a mixing coil 75 in order to achieve the coloring and color development of the

. To effect eosinophils in the blood surge on a selective basis. The reaction given in the above equation applies to the color development, with the exception that the pH is 3.5 and room temperature prevails. Following the color development, the refractive index of the red cell walls is adjusted, for example by adding propylene glycol, which is supplied via a pump hose 77 and, after being introduced into the partial flow, is mixed with it in a mixing coil 79. The oil stream coming from the mixing coil 79, which contains the selectively colored eosinophils, is passed through a

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Line 81 is fed to a flow cell arrangement 83

The remaining analysis channels AIII and AIV of the overall arrangement are used for the selective staining of the basophils or monocytes. The partial flow supplied via the pump hose 17 is combined in the analysis channel AIII with a flow which contains suitable coloring and lysing agents, for example 0.1 $> neutral red and 1 $ saponin, buffered to a pH of "about 5.5. This The stain and lysing agent stream is supplied via a pump hose 85 and is divided by air pulses which are introduced via a pump hose 87. The combined streams are completely mixed in a mixing coil 89. The erythrocytes are dissolved and the staining of the leukocytes is started The partial flow coming from the mixing coil 89 is combined with a flow of formalin which is supplied via a pump hose 91. Subsequently, mixing takes place in a mixing coil 93 in order to fix the leukocytes in the partial flow Partial stream, which contains selectively colored Basopbile, is with a stream of propylene glycol col, which is fed via a pump hose 95 in order to adapt the refractive indices in the actual partial flow. From a mixing coil 97, the partial flow treated in this way passes via a line 99 to a flow cell assembly 101. The chemical procedure for the selective staining of basophils in a whole blood sample is known as the "basophil neutral red method" and is described on pages 1126 to 1127 of the above mentioned book by John B. Miale, M ₁ D ,,.

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Finally, the monocytes are selectively stained in the analysis channel AIV, as described, for example, in the patent application filed at the same time. As shown, the partial flow supplied via the pump hose 19 is first combined with a flow of fixative, for example 20 # formalin, buffered to a pH of about 6 with a 0.1 molar phosphate buffer. This formalin flow is fed in via a pump hose 103 and, before being merged with the partial flow, is divided by air thrusts which are fed in via a pump hose 105. The "partial flow and the fixative flow are brought together in a cooling bath 107, the temperature of which is, for example, 4 degrees Celsius _{T in} order to prevent any chemical reaction between the cells or the plasma and the fixative until in a mixing coil 109, which is also located in the The flow coming from the cooling bath 107 is brought together with a substrate, for example with caphtol butyrate. The substrate is supplied via a pump hose and mixed with the partial flow in a mixing coil 113. The leukocytes are mixed The fixed partial flow is brought together behind the mixing coil 113 with a flow of a coupling reagent, for example of hexazonium-pararosaniline Monocyte cause selective staining and color development in successive batches of blood samples. The following equation applies to color development:

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^"19 ~ 21534Θ5

Lipaee in monocytes a) -aphthalene butyrate naphtol

pH 6.1, room temperature

b) -Napbtol + hexazonium pararosaniline ——— colored

Precipitation

The partial flow coming from the mixing coil 117, the contains selectively stained monocytes, i is then combined with a stream of acetic acid which is passed over a pump tube 119 is supplied to lyse the erythrocytes. The erythrocyte lysis is going on accelerated by the fact that the partial flow is passed through a mixing coil 121. Behind the mixing line 121 the partial flow with the selectively stained monocytes is combined with a flow of propylene glycol, the is introduced via a pump hose-123. Through this are said to be the refractive indices of the current and the cell walls of the lysed erythrocytes are adapted to each other · For this purpose, the partial flow is passed through a mixing coil 125 led. From there the partial flow arrives via a Line 127 to a flow cell arrangement 129

The flow cell assemblies 65, 831 101 and 129 assigned to the analysis channels I, II, III and IV are preferably constructed in such a way that the partial flow of the relevant analysis channel starts from a sheath flow surrounded by an inert liquid and carried along by this sheath flow. This reduces the coaxial the partial flow aligned with the sheath flow is the diameter of the partial flow. In this way it is achieved that the leukocytes present in the partial flow are concentrated on a small area and on

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in this way flow through the viewing chamber of the flow cell arrangement practically one behind the other. The den Partial flow surrounding liquid sheath flow prevents at the same time that the viewing chamber through any granular Particle is blocked. The partial flow is preferably compressed to a diameter of approximately 75 micrometers. This practically eliminates the risk that two or more leukocytes next to each other the viewing chamber 151 flow through. The diameter of the sheath stream is about 250 micrometers. This ensures that any artifacts or granular particles do not clog the viewing chamber 151.

The flow cell assemblies 65 »83, 101 and 129 and the associated photometric devices have the same structure, so that the corresponding parts are provided with the same reference numerals. The photometric associated with each flow cell assembly The device detects the passage of the colored leukocytes through an absorption process, for example to count the colored leukocytes passing through the light chamber 151, but also serves for detection of the colored and unstained leukocytes flowing through the viewing chamber 151 according to a scattering method. The scattered signals obtained according to the scattering method are used for a volume calculation method, with the Analysis results of the individual analysis channels are related to one another, and are also used to provide control signals for the logic arrangement.

Durohm flow cell assemblies 65, 83, 101 and 129 each contain a Duroh Flussküvette 153, which is connected to the

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respectively assigned power 63, 81, 99 or 127 is connected to take up the colored and lysed partial flow of the analysis channels I, II, III or IV · In the lines 63, 81, 99 and 127 a degassing device 155 is arranged in each case, which the lines to the atmosphere ventilates · All the electricity. separating air bubbles, namely both the air thrusts located between the individual blood samples and the additional air thrusts arranged within a sample thrust, so that the stream only consists of liquids that are fed to the flow cells 153 via lines 157. After the venting, the partial flow flowing through the duct 157 thus only contains adjacent blood samples, which are each separated from one another only by a surge of washing liquid. The thrust of the washing liquid prevents the following sample cubes from being contaminated by the previous one as it flows through the flow-through cuvette 153. The line 157 ends in a first tubular inlet, the end of which is arranged concentrically with respect to the viewing chamber 151 of the flow cell. The jacket liquid surrounding the partial flow is supplied from a pressurized storage container 159 which is kept at a constant positive pressure by a pump 161 and an associated pressure regulator 163. The outlet pipe from the lug-shaped. Storage container 159 is connected to a distributor line 165 which leads to the flow cells 153 via an adjustable flow control valve 167. The sheath flow liquid coming from the flow valve 167 is fed to the flow cell 153 via a second tubular inlet 169. The tubular inlet 169 is arranged and designed so that the supplied

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Jacket liquid surrounds the partial flow concentrically and takes him to viewing chamber 151. '

Since the AI, All, AIII and AIV assigned to each analysis channel hydraulic system is open, namely that each partial flow at the degassing device 155 is vented, the partial flow and the bypass flow are maintained by a liquid drain tank 171 constant negative pressure drawn through flow cell 153. To this end, the Liquid drainage tank 171 is connected to a vacuum pump 173 through a vacuum regulator 175. As shown, the outlet of each flow cell 153 is connected to the drain tank 171 via a line 177. Any line 177 contains a resistance coil 179 with a high flow resistance. The resistance snakes are respectively used for temperature compensation in a heating bath 181, around the flow through the flow cell 153 against smaller fluctuations in the flow resistance in the stabilize hydraulic system and ensure that in the flow cells 153 a constant flow is maintained. The use of temperature compensated Resistance snake with high flow resistance in continuous flow arrangements has been proposed.

The substreams treated in the analysis channels AI, All, AIII and AIV are sent through the assigned flow-through cells 153 in a suitable phase relationship to one another. Although a device for the independent adjustment of the relative phases of the partial currents is not shown, the device known from US Pat. No. 3,512,163 can be used for this purpose.

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The leukocytes of the substreams passing through the viewing chamber 151 are detected simultaneously by the scattering method and the absorption method. The equipment required for this is only shown schematically, since such devices are generally known. In this regard, reference is made to the following literature: "Measurement of Small Particles Using Light-Scattering: A Survey of the Current State of the Art", AE Martens, Ann. NY Acad. May be. 558: 690-702; "Rapid Multiple Mass Constituent Analysis of Biological Cells," Kamentsky, LA, et al., Ann, NY Acad. May be. 157: 310-323; and "Absorbtion Cytophotometry: Comparative Methodology for Heterogeneous Objects, and the Two-Wavelength Method" ML Mendelsohn, in "Introduction to Quantitative Cytochemistry ¹¹ , edited by George L. Wied, Academic Press 1966, pages 201-214. This is shown Light emitted by a light source 183 is collimated by a condenser lens arrangement 185 and sent through a diaphragm 169 provided in a diaphragm plate 187 in order to irradiate the concentrically restricted partial flow which flows through the viewing chamber 151. The actual viewing volume in which the leukocytes are detected, is essentially determined by the diameter of the partial flow in the viewing chamber 151 and the dimensions of the diaphragm 189. The dimensions of the diaphragm 189 in the flow direction of the partial flow should preferably be minimal , but in relation to the size of the cells to be measured and the flow of the partial flow in one stand such a ratio that the cuvette is not clogged or clogged r the response and sensitivity range of the scatter and / or absorption measuring device can be limited. Furthermore, the dimensions of the diaphragm 185 running transversely to the direction of flow of the partial flow V should include the total diameter of the partial flow and also any

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Consider fluctuations or vibrations in the course of the flow. The light beam passing through the viewing chamber 151 is thus from each individual leukocyte in the Partial flow "affected, regardless of whether the leukocyte is colored or not. The light beam reaches a beam splitter 189 from the viewing chamber 151. The one light beam leading away from the beam splitter 189 passes through a light beam provided in a diaphragm mask 193 Aperture 191 and impinges on a photodiode 195 to the light absorption caused by each colored leukocyte to prove. The second beam from beam splitter 189 is directed to a photomultiplier tube 197 directed, but in the beam path an aperture mask 199 is arranged with an annular aperture, so that only that of the colored light scattered and non-stained leukocytes of the substream strikes the multiplier tube 197.

The ones from the photodiode 195 and the photomultiplier 197 generated absorption and scatter signals are fed to logic channels LI, LII, LIII and LIV, assigned to the analysis channels AI, All, AIII and AIV are. In this way it is made possible to identify the individual populations of the various sub-streams automatically determine existing leukocytes and correlate them with respect to the same blood sample.

In order to explain the logic operations for determining the populations of the individual leukocytes, first reference is made to the logic channels LII and LIV associated with the analysis channels All and AIV in order to the populations of eosinophils and monocytes increase counting. The logic channels LI and LIII, which are assigned to the analysis channels AI and AIII, show apart

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basically the same structure with some additional logic units. As mentioned earlier, the logic channel LI is used to determine the population of neutrophils that have been stained together with the eosinophils in a group and the population of lymphocytes in the blood sample. For the sake of simplicity, similar parts in the logic channels LI to LIV are provided with the same reference numerals · ¹

As can be seen, in the logic channels LII and LIV, which are assigned to the analysis channels All and AIV, the scatter signals generated by the photoelectron multiplier 197 when colored or unstained leukocytes pass through the viewing chamber 151, and the absorption signals generated during Passage of colored leukocytes through the viewing chamber 151 is generated by the photodiode 195, amplified by amplifiers 201 and 203, respectively. The amplifiers 201 and 203 are followed by comparators 205 and 207, respectively, which work as threshold value devices in order to block any noise signals that may occur in the scatter and absorption signals. The outputs of the comparators 205 and 207 are connected to the inputs of monostable MuVbivibrators 209 and 211, respectively. The output pulses generated simultaneously by the monostable multivibrators 209 and 211 correspond to the temporal occurrence of a colored leukocyte in the viewing chamber 151 of the associated flow cell 153. If, on the other hand, the monostable multivibrator 209 alone emits an output signal, this corresponds to the temporal occurrence of an uncolored leukocyte in the viewing chamber 151 of the Duroh Flussküvette 153.

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The output of the monostable multivibrator 211 is connected to one input of an AND element 213, while the output of the monostable multivibrator 209 to the other input of the AND gate 213 and to the one input of an AND gate 215 is connected. The output of the AND gate 213 is with the input of a AND gate 217 connected. The other inputs of the two UHD elements 215 and 217 are connected to the set output a flip-flop 219 is connected. The flip-flop 219 becomes

^ only set if a previous counting process has ended and has been printed out by a printer 221 and a next counting process is to be initiated. For this purpose, the set input of the flip-flop 219 is connected to the output of an AND element 223. Of the One input of the AND element 223 is connected to the printer 221 via a line 225. After finishing each During the printing process, the printer 221 applies a signal to the line 225. The other input of the AND gate 223 is connected to the output of an adjustable clock generator 229 via a line 227. The clock 229 speaks in each case on the indexing of the turntable 5 in order to apply a control pulse to the line 227, the

is delayed in such a way that it occurs with the flow of the treated partial flows of the sucked blood sample through the viewing chamber 151 of the various associated analysis channels collapses. The clock 229 thus delivers a series of control pulses that begin with the passage of successive blood samples through the Flow cells 153 are in phase.

Assuming that the analysis results of a previous Counting operation have been printed out by printer 221 and a signal is present on line 225 indicating that To prepare AND gate 223, turns on the clock

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229 via the line 227 incoming pulse through the AND gate 225, so that the flip-flop 219 is set. When flip-flop 219 is set,. there is a signal at the AND gates 215 and 217. The outputs of the AND gates 215 and 217 are connected to the inputs of a preset counter 231 and a balancing counter 233, which "have already been reset to zero by a reset pulse that was issued by the printer 221 at the end of the previous printing operation via a line 235 The AND gates 215 and 217 thus control the operation of the preset counter 231 and the balancing counter 233 such that the associated populations of the colored leukocytes which are colored in each of the substreams and are present in the same original blood sample, The scatter pulses generated by the multivibrator 209 in the basic logic arrangement thus run through the AND gate 215 to the preset counter 231, the count of which indicates the total number of leukocytes, i.e. the colored and non-colored leukocytes that since the beginning of de r counting operation, that is, since the flip-flop 223 was set, »have flowed through the viewing chamber 151 of the associated flow-through cuvette 153. At the same time, the absorption pulses generated by the multivibrator 211 in the basic logic arrangement arrive via the AND element 213 and the AND element 217 to the balancing counter 233, the count of which indicates the total number of colored leukocytes from the total count of the preset counter 231. The percentage population of the stained leukocytes that are counted by any logic channel can thus be given immediately by using the ratio

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between the relevant count value of the preset counter 251 and the count value of the balance counter 233 forms. The larger the count value of the preset counter 231, the more accurate the statistical fall Determination of the population of the relevant leukocytes.

As a result of the operation of the AND gate 213, the balance counter 233 only counts an absorption pulse when. a stray pulse occurs at the same time. The AND element 213 therefore scans the absorption signals in order to thereby switch off any incorrect counting pulses which could occur due to random large noise signals at the output of the absorption amplifier 203 and which arise, for example, from artifacts flowing through the assigned viewing chamber 151 or generating noise signals in the amplifier whose amount is greater than the threshold value of the comparator 207 and which thus reach the multivibrator 211. However, it is possible that when a large noise signal occurs simultaneously in the absorption channel and a stray pulse from the multivibrator 209 due to the presence of an unstained Iieukocyte in the viewing chamber 151, a pulse passes through the AND gates 213 and 217 and is counted by the balance counter 233 will. Statistically, however, this happens very rarely. The ratio between the assigned count values in the balancing counter 233 and the preset counter 233 consequently provides a very precise result for the percentage population of the selectively stained leukocytes in the relevant analysis channel. The preset counter 231 is preferably limited to a predetermined count, for example 10,000 or any multiple of 10O ₉ , while the balance counter 233

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is not limited. When the default counter 231 is Counting ended, for example at 10,000, it prevents the associated balance counter 233 from continuing counts. For this purpose, the default counter 231 delivers a control pulse via a line 237 to the reset input of the flip-flop 219. The reset flip-flop 219 blocks the AND gates 215 and 217 and consequently prevents the default counter 231 and the balance counter Count 233 further scatter and absorption impulses. Farther is the output of the default counter 231 via the Line 237 is connected to an associated input of an AND gate 239 which enables the operation of a multiplexer 241 initiates. The other inputs of the AND gate 239 are connected to the corresponding parts in the other logic channels connected. This will, when the logic and counting operations are completed in all channels, an indication is provided indicating that printer 221 can output information. The UHD element 239 is delayed therefore the output of the information collected in the individual logic channels LI, LII, LIII and LIV as long as until the logic and counting operations relating to a particular blood sample are completed in all of these channels are. Only then is it possible to correlate the information and with regard to a particular blood sample, that is of a special blood donor, to print out.

The machine function of this logic channel can be monitored by a speed monitor 243 and a pen writer 245. The input of the speed monitor 243 is connected to the output of the multivibrator 209. The output of the speed monitor 243 is connected to the pen drive mechanism of the recorder 245. The speed monitor 243 integrates the stray pulses generated by the multivibrator 209 and controls the deflection of the pen. The distraction dee

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Pen in pen 245 delivers a continuous Display of the concentration of leukocytes flowing through the viewing chamber of the flow cell 153. The leuko— cyte concentration in each viewing chamber 151 changes due to the presence of a surge of wash fluid between two consecutive blood samples in the Partial flow and, since the air segments have been discharged are, also as a result of a certain mixing of the washing liquid thrust, .with the adjacent blood samples. When a blood sample with a constant dilution through the viewing chamber 151 of the associated flow cell 153 flows, the deflection of the pen is practically flat. So it will be a steady state displayed. Furthermore, the control pulses generated by the clock generator 229 are sent to the speed monitor via the line 227 supplied so that the writing trace recorded by the pen is superimposed with a blip indicating the beginning of a counting operation. At the same time, the flip-flop 223 is set so that the Scatter and absorption pulses can reach the default or balancing counter 231 and 233, respectively. If this Blip does not coincide with the plateau of the recording track, then it means that the counting operation in relation to the flow of the blood sample in the partial flow through the associated analysis channel and the associated flow cell 153 is not timed. In this pail one can see the phase or expiry time of the partial flows in relation to the assigned Adjust flow cuvettes 153 individually with devices known per se, which are described, for example, in US Pat 3 512 163 are described '

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The above-mentioned logic operations can be used when a single type of leukocyte is colored in a special Analysierkanal and their percentage population to be determined. The logic channels LII and LIV are suitable for determining the percentage populations of eosinophils and monocytes in the total count of the assigned default counter 231. When the counting operation in each logic channel has been completed, the particular count of the stained leukocytes remains in the associated balance counter 233 and is available for recording. Each logic channel is blocked by the operation of the assigned default counter 231 because the plip-flop 219 is reset. This blocks AND gates 215 and 217.

The logic channels LI and LIII basically contain the same logic arrangement. The logic channel LI, however, contains additional logic circuits, on the one hand to selectively differentiate between the neutrophils and the total number of neutrophils and eosinophils, which are selectively colored as a group in the analysis channel AI and are also counted as a group in the assigned balancing counter 233, and on the other hand for counting of the lymphocytes, which are not stained in the analysis channel AI, but can be determined due to their size by a pulse height differentiation of the scattered pulses. Additional logic circuits are provided in the logic channel LIII in order to selectively and necessarily spectrally distinguish the basophils that are selectively colored in the analysis channel AIII from noise signals which have no particular spectral profile. In the logic channels LI and LIII, similar parts of the basic logic arrangement are provided with the same reference numerals.

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As described so far, the multivibrator 211 also emits absorption pulses in the case of the logic arrangement IfI starting if either an eosinophil or a neutrophil through the viewing chamber 131 of the associated analysis channel AI run. The count of the balance counter 233 therefore gives the total population of these leukocytes from the count of the default counter 231 of the logic arrangement LI at. As already described above, the count of the balance counter 233 of the logic channel LII gives the total. number of eosinophils from the count of the associated "Yorgaberegister 231" in the associated batch thereof Blood sample in the analysis channel All on. The default counters 231 in the logic channel LI and LII are the same Count value limited. The outputs of the balancing registers 233 of the logic channel LI and the logic channel LII are connected to the inputs of a subtraction register 247. Furthermore, the subtraction register is 247 connected to the output of the ÜHD member 249, whose Inputs are connected to the lines' 237, which lead to the default counters 231 in the logic channels LI and LII to lead. If the counting operation has thus been terminated in both logic channels LI and LII, the AND element is 249 frees the subtraction register 247 to carry out the subtraction. The subtraction register 247 subtracts the count of the eosinophils in the balance register 233 of the logic channel LII of the total count of the eosinophils and neutrophils in the balance register 233 of the logic channel LI. The resulting difference, which indicates the number of heutrophils in a particular blood sample, will be saved. When the subtraction operation is completed, the subtraction register 247 asserts a signal one input of an AND gate 251 »the other Input already via a line 253 a signal from the default counter 231 in the logic channel LI receives. With

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switching through the AND gate 251 indicates that the logic arrangements LI and LII and the subtraction register 247 have completed their operations. The AND element 251 again applies an input signal to the AND gate 239. Since the gate counter 231 in the logic arrangements LI and LII are limited to the same count, the total count of eosinophils is im Balance counter 233 of logic channel LII and the total count of neutrophils in subtraction register 247. in proper relationship with respect to the same blood sample.

Furthermore, the logic arrangement LI contains additional logic devices to the percentage population of the Lymphocytes in the blood sample by a pulse height discrimination method to determine. Since the lymphocytes and monocytes are agranulocytes, they are not stained, as is the case with the eosinophils and neutrophils in the analysis channel AI of the Pail. The lymphocytes can can easily be distinguished from monocytes by that they have a differently sized cell structure. In general, a lymphocyte has an essential smaller diameter, for example of about 7 micrometers, than a monocyte whose diameter is about 12-20 microns. Since the amount of light scattered increases with the size of the cell structure, it is possible to determine the presence of a lymphocyte in the viewing channel 151 of the analysis channel AI. To this end is the output of the leakage pulse amplifier 201 of the logic arrangement Lli via a line 257 to a second comparator 255 connected. The comparator 255 only provides an output signal if the amplified scatter signal exceeds the amount generated by a lymphocyte flowing through the viewing chamber 151. The exit dee

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Comparator 255 is connected via a line 259 to a multivibrator 261, the output of which is connected to one input S ^ of an anticoincidence element 263. The outputs of the scattering multivibrator 209 and absorption multivibrator 211 of the logic arrangement LI are connected to the second input S ^ and the third input A of the anticoincidence element 263. Since the pulses fed to the inputs S 1, S _H and A of the anticoincidence element 263 are due to the presence of the same leukocyte in the viewing chamber 151 of the analysis channel AI, these pulses occur simultaneously. The anticoincidence member 263 generates the function S- ^ S ^ 'A. The absence of a signal at input A thus indicates that the particular leukocyte in the viewing chamber 151 is not colored, that is to say it is an agranulocyte. The presence of a signal at the input S " indicates that the leukocyte in the viewing chamber has a size that exceeds the normal size of a lymphocyte, ie that it is a monocyte. The presence of a signal at the input Sj ₁ means that there is a leukocyte in the eye chamber. The anticoincidence member 263 thus detects a lymphocyte in the viewing chamber 151 of the analysis channel AI.

The output of the anti-coincidence element 263 is connected to one input of a second blocking MD element 265, the other input of which is connected to the set output of the flip-flop 219 in the logic channel LI. The output of the UHD element 265 is connected to the input of a second balancing counter 267, the count value of which indicates the number of lymphocytes from the total number of the default counter 231 of the logic arrangement LI. The output of the balancing register 267 is connected to an associated connection of the multiplexer 241. The mode of operation of the balance counter 267 therefore corresponds to the mode of operation of the

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Balance counter 233 in the logic arrangement LI similar, that it is prevented from continuing counting when the dip-flop 219 is reset by the printer 221. Also will continue the balance counter 267 by a control pulse on the line 235 after the completion of printing by the Printer reset. Since the count value of the balance counter 267 is based on the count value of the default counter 231 of the logic channel LI is related, with the switching through of the AND element 241, for the purpose of controlling the UMD element 239 » indicated that the lymphocyte count is available stands.

Regarding the counting of the basophils, their population generally less than 1 # of the total white blood cell population in a blood sample, the logic arrangement LIII is constructed in such a way that it possibly artefacts that occur, for example dust particles, gas bubbles, etc., caused by the associated Viewing chamber 151 flow. Furthermore, random electrical noise signals are taken into account drawn, which cause a false count and thus the determination of the percentage population of the Adversely affect basophils in the blood sample could. For this purpose, the beam splitter 189 in the flow cell arrangement 101 is a two-color one Associated mirror, which reflects the blue portion of the absorption signal to a photodiode 271 and the red component to a photodiode 273 passes. A diaphragm plate 193 of the type described is arranged in front of the photodiodes 271 and 273. The logic channel LIII therefore contains parallel absorption channels for processing the red and blue components of the Absorption signal. Since the basophils are colored red,

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absorb blue light. One in the viewing chamber 151 of the analysis channel AIII present colored Basopb.il therefore reduces the intensity of the Falling light photodiode 271. However, the presence of the colored basophil does not decrease those Intensity of the light falling on the photodiode 273, In general, EaIl is therefore the signal of the smallest amplitude, which is due to the presence of a colored Basophils is generated in the viewing chamber 151 of the analysis channel AIII, during a counting sequence at the -t output the photodiode 273 occur. Instead of providing dedicated logic to distinguish these signals, logic channel LIII counts the basophils and all noise pulses as well as the noise pulses individually and then subtracts these counts by the percentage Indicate the population of basophils in the particular blood sample. >

The output of the photodiode 273 is to an amplifier 275 connected, the output signal of which is fed to the input of a comparator 277. The comparator 277 compares the output of amplifier 275 and only provides an output signal if the amplitude of the amplified signal is the same as the amplitude of one Signal that indicates the presence of a colored basophil in the viewing chamber 151. The comparator 277 does not emit an output signal when a basophil is present in the viewing chamber 151. The output signal of the comparator 277 thus means that a noise signal has occurred which is incorrectly identified as Sasophil could be counted. The parallel comparator 207 in the logic channel LIII is via the amplifier 203 on connected to the output of the photodiode 271. This comparator 207 provides an output signal when the

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Signal of the amplifier 203 a predetermined threshold value exceeds. The output signal of the comparator 207 in the logic channel LIII thus indicates the presence of a sasophile in viewing chamber 151 or the presence of a noise signal that is incorrectly could be counted as a Sasophil. The outputs of the comparators 207 and 277 are to the inputs of multivibrators 211 or 279 connected. The multivibrator 211 thus generates an output pulse when a basophil Absorption signal or a large noise signal occur. The multivibrator 279, however, only generates then an output signal when a large noise signal occurs. The outputs of the multivibrators 211 and are connected to one of the inputs of the AND gate 213 and an AND gate 281. The other entrances to the AND gates 213 and 281 are connected together to the output of the multivibrator 209 in the logic channel LIII. These AND gates are thus prepared by a scatter signal. The outputs of the multivibrator 209 and the AND gates 213 and 281 each lead to an input of the blocking AND gates 215, 217 and 283 in logic channel LIII. The other inputs of these AND gates 215, 217 and 283 are connected to the set output of the flip-flop 219 connected. When the flip-flop 219 is set and the blocking AND gates 215, 217 and 283 are prepared during a counting cycle, the stray pulses generated by the multivibrator 209 become counted by the default counter 231, while the balance counters 231 and 285 are simultaneously with these Scattered pulses count absorption pulses that are generated by the multivibrators 211 and 279.

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The outputs of the accumulation registers 233 and 285 of the logic channel LIII are connected to the inputs of a subtraction register 287 connected. When the counting cycle in logic channel LIII is finished, what is caused by the default counting of the default counter 231 is indicated, the default counter releases the subtraction register 287 a signal on a line 289, so that the subtraction register the count values of the balance counter 233 and subtracts 285 and stores the difference, which is the total number of basophils that responded to that of the default register 231 of the logic channel LIII specified total number of leukocytes fall. When the subtraction register has finished its operation, it controls the an input of an AND gate 291, the other input of which has already been previously determined by the default counter 231 of the Logic channel LIII is controlled. The output of the AND gate 291 is connected to an input of the AND gate 239. If a signal is applied to this input, this means that the count of the basophils in the logic channel LIII has ended.

After each counting operation has ended, the count of all lymphocytes is thus in the balance counter 267 of the logic channel LI saved. The total neutrophil count is located in the subtraction register 247, which responds to the balance counters 233 in the logic channels LI and LII. The total count of the eosinophils is stored in the balance counter 233 of the logic channel LII. Of the Count of all basophils, is in the subtraction register 287, which is on the Saläiercounters 233 and 285 of the logic channel LIII responds. The total count of the monocytes is finally in the balance counter 233 of the

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Logic channel LIV saved. You outputs this counter and registers are connected to the inputs of multiplexer 241 as shown. The percentage The total population of the individual cell types is determined by the The ratio of the assigned count to the count of the assigned default counter 231 is given. It is It is advantageous if the default counters 231 of the individual logic channels LI, LII, LIII and LIV have the same count value be limited, preferably at 10,000, so that the percentage population of each cell type directly through the two most significant digits of the individual ones contained in the balance counters and registers Values are given · This eliminates the need for additional circuits forming the ratio.

The multiplexer 241 operates in such a way that it reads at least the two most significant digits of each counter or Reads registers preferably one after the other. When the counting sequence is finished, 'are present at all inputs of the UMD element 239 signals. Hence the UKD link 239 sends a trigger pulse to the multiplexer 241 via a line 293. As a result, the information of the Balance counter 267 of the logic channel LI (lymphocytes), the subtraction register 247 (neutrophils), des Balance counter 233 of the logic channel LII (eosinophils), the subtraction register 287 (basophils) and the The balance register 233 of the logic channel LIV (monocytes) is successively supplied to the printer 221. Of the Printer 221 continuously prints out the percentage populations of each of the different cell types, which are contained in the same blood sample and how they are correlated from the relevant counters and registers in ex Way to be specified. When the multiplexer 241

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21 5340S

its scanning and the printer 221 has completed its output operation, the printer 221 generates a control pulse which appears on the line 235 in order to combine the]? lipflops219 and the default counters 231 as well as the balancing counters 233 in the logic channels LI, LII, LIII and LIV with the other balance counters and 285 in the logic channels LI and LIII. When the next control pulse then occurs in line 227, which indicates that the colored bursts of the next blood sample in the partial flows flow through the viewing chambers 151 of the analysis channels AI, All, AIII and AIV, the AND gates 223 are switched through and thus the flip-flops 219 in all logic channels are set to begin the counting operation for the next blood sample.

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Claims (47)

Claims f 1J device for determining the percentage a selected particle type within a group of particle types contained in a medium, characterized in that a treatment device treats the medium in such a way that the properties of the selected particle type change, while the corresponding properties the other types of particles remain practically unchanged that a first device within a reference measure counts the particles occurring in the medium, the properties either changed or unchanged have that a second device selects the particles on the basis of the changed properties discriminated and counts the selected particles of the medium within the reference measure, and that a device relates the counts of the first and second counts to the percentage of the selected particle type to be specified in the medium. 2. Device according to claim 1,
characterized in that the medium is a liquid and a device is provided which passes this liquid through a viewing chamber, and in that the first and second devices count the particles with the changed properties and the particles with the changed or unchanged properties, respectively, when they durob the sight chamber flow. ../42 209821/0884 3. Device according to claim 2,
characterized in that the treatment device changes the spectral properties of the selected particles, that the first device contains a device which detects the selected particles on the basis of the changed spectral properties, and in that the second device contains a device which, on the basis of one of the changed and unchanged particles common property detected the passage of the particles with changed or unchanged properties through the viewing chamber. 4.Vorrichtung according to claim 3,
characterized in that the second device contains a photometric device which detects the passage of the particles with changed and unchanged properties through the viewing chamber. 5. Device according to claim 4 »
characterized in that means sends a beam of light through the viewing chamber and in that the photometric device is responsive to the beam of light sent through the viewing chamber. ../43 209821/0884 6. Apparatus according to claim 2,
characterized in that the first and the second device respond to particles which pass through the same section of the viewing chamber, and that the first and the second device simultaneously count particles with changed properties and the particles with changed or unchanged properties. 7. Apparatus according to claim 6,
characterized in that the second device is limited to a predetermined count and includes a device which locks the first device when the predetermined count is reached in the second device, so that the count of the first device represents the proportion of the selected particles within the specified count value. 8. Apparatus according to claim 7 _f
characterized in that the second device is limited to a count which is a multiple of one hundred. 9. Apparatus according to claim 7 _f
characterized in that the second device is limited to a count of 10,000, the first two most significant digits of the count of the first device, when it is disabled, indicating the percentage of the selected particles in the medium, ../44 209821/0884 2153A05 10. Apparatus according to claim 7,
characterized in that means responsive to the first and second means records the ratio of the counts of the first and second means. 11. The device according to claim 7,
characterized in that the recording means is responsive to the first means for recording the number of selected particles counted by the first means. 12. A device according to claim 1. A _n,
characterized in that a device guides the medium through a viewing chamber, that a photometric device is arranged at the viewing chamber, which detects the passage of particles with changed or unchanged properties through the viewing chamber and which discriminates the passage of particles with changed properties, that the photometric means including first and second means for providing an indication of the detection or discrimination, respectively, of the particles passed through the viewing chamber, and first and second counter means counting the indications provided by the first and second means, respectively, the second Counter device is designed such that it counts the displays supplied by the second device only in the event of coincidence with a display supplied by the first device to the first counter device. ../45 209821/0884 13. The device according to claim 12, characterized in that that a gate device connects the second device to the second counter device and that the Gate means responsive to said first means, said second counter means upon coincidence the first display from the first device with the second display from the second device a count display undertakes. 14. Apparatus according to claim 2,
characterized in that negative pressure pressure means directs the liquid through the viewing chamber. 15. The apparatus according to claim 14, characterized in that the printing device is a resistance coil with a high flow resistance, the sioh between the viewing chamber and the Druckeinriohtung is located. ../46 209821/0884 16. The device according to claim 1, characterized in that that the medium is a liquid sample, that a device divides the liquid sample into several partial flows divides and treats each! bearing stream individually to determine the properties of selected particles in each of the Partial flows change, while the properties of other particles in each of the partial flows remain practically unchanged remain that a first device assigned to each partial flow in the assigned partial flow the Particles with either changed or unchanged properties within one of the relevant substreams assigned reference measures detected and counts that a second device assigned to each partial flow the selected Particles in the assigned partial flow are discriminated on the basis of the changed properties and the selected Counts particles in the assigned partial flow within the same assigned reference dimension, and that a device the respective counts of the first and second device, which the respective Partial streams are assigned, related to each other, to the percentage of selected Determine particles in the liquid sample. 17. The device according to claim 16, characterized in that that a device creates the same reference measure for each of the substreams, so that. the percentages of all selected particles in the liquid sample are related to each other. ../47 209821/0884 18. The device according to claim 2, characterized in that a device has several successive Liquid samples in the form of a continuous stream that a device supplies this stream divided into several sub-streams and each sub-stream treated individually in order to determine the properties of each sub-stream of selected particles to change so that the selected particles of other particles can be discriminated or differentiated in the same substream that the device manufactured Particles with either changed or unchanged properties are detected in each of the substreams and counts that the second device, the selected particles in each of the substreams due to the changed Properties discriminates and counts that a device counts the first and second Establishment in relation to each of the partial flows relates to each other to the percentage To determine the proportion of the selected particles in the feathers of the partial flows, and that a device that recorded percentages in relation to the sample in question in a correlated manner. 19. The device according to claim 18, characterized in that that a device timed each of the substreams through a respective assigned viewing chamber directs that an associated first and second device are each associated with one of the viewing chambers are and work in relation to flede viewing chamber simultaneously and that the recording device on the first and second means associated with each viewing chamber are responsive to correlate with respect to the liquid sample Way to record the percentage. 209821/0884 ../48 2153A05 20. Apparatus according to claim 18, characterized in that that the first and second devices, each associated with a viewing chamber, are selected Count particles or the particles with changed or not changed properties within the same reference dimension. 21. The device according to claim 18, characterized in that that the first and second means, each associated with a viewing chamber, with respect to a predetermined count of the particles with changed and unchanged properties for each Partial stream specify a count of the selected particles. * 22. The apparatus according to claim 18, characterized in that each of the second devices, each one the viewing chambers are assigned, is limited to the same predetermined count and that the respectively assigned first device during the counting process of the second device up to the predetermined count value counts the selected particles. ../49 209821/0884 23. The device according to claim 18, characterized! that the treatment facility has the spectral properties shafts of the selected particles in each substream changes and that the second device changes the selected Particles in each partial flow due to the discriminated against changed spectral properties. 24. Device for determining the percentage of a selected type of leukocyte in a blood sample , characterized in that a treatment device takes the blood sample in such a way treats that at least one selected species of leukocyte is selectively stained, that an observation device observed at least a portion of the treated sample to discriminate the stained leukocytes and to detect and also the stained and unstained leukocytes in that part of the sample to detect that a first means detects the colored leukocytes in said portion of the blood sample counts to provide a first count and that a second means the detected colored and counts unstained leukocytes in said part of the blood sample to provide a second count, wherein the ratio of the first and second counts is the percentage of the selected Specifies the type of leuiocyte in the blood sample. ../5O 209821/0884 25. Gate direction according to claim 24, characterized in that that the second counting device is limited to a predetermined number and that one device is limited to the second counter responds to disable the first counter when the second counter Ms counted to the given number. 26. Door direction according to claim 24, characterized in that that a device determines the ratio between the first and the second count of the first and second counting device records. 27. Gate direction according to claim 24, . characterized in that the first counter is operated only simultaneously with the operation of the second counter. 28. Gate direction according to claim 24, . characterized, * that the detection device for the detection of the colored Leukocytes containing a photometric device. 29. Gate direction according to claim 24 »characterized in that the treated blood samples are passed through a viewing chamber and that the viewing chamber is assigned a photo-r metric device which discriminates and detects the colored leukocytes and which detects the colored and non-colored leukocytes pass through at least a portion of the viewing chamber, the first and second counting means being responsive to the pbotometrlsohe means. 209821/0884 ../51 30. The device according to claim 29, characterized in that a comparator device connects the photometric device and the first and the second counting device. 31. The device according to claim 29, characterized in that the photometric device is a light beam through the part of the viewing chamber that sends a first Device detects the absorption of the light beam in order to indicate the passage of the colored leukocytes, and that a second device detects the scattering of the light beam in order to prevent the passage of colored and View unstained leukocytes through the part of the viewing chamber, showing the selectively stained leukocytes are discriminated and the first and the second counting device on the first and second detection device speak to. 32. Apparatus according to claim 31, characterized in that the first counting device is actuated by the first and second detection device upon simultaneous detection of absorption and scattering of the light beam. ../52 209821/0884 33. Apparatus according to claim 31, characterized in that the first detection device for generating a first pulse contains a first pulse device and the second detection device for generating a second pulse contains a second pulse device that, when a first and a second pulse occur simultaneously, the first counting device is actuated for counting the number of passing through the! part of the viewing chamber stained leukocytes indicate that the second counter counts the second pulses to the total number of leukocytes indicate, that the colored and non-colored leukocytes duroh the rope Pass through the viewing chamber. 34. Apparatus according to claim 33, characterized in that a first gate device connects the first and the second pulse device to the first counting device. 35. Apparatus according to claim 34, characterized in that the second counting device is limited to a predetermined count and that when the predetermined count is reached, a device responds to the second counting device in order to prevent the first counting device from continuing to count, so that the count in the first counting device indicates the total number of selected leukocytes within the specified number of leukocytes, ../53 209821/0884 36. Apparatus according to claim 35, characterized in that that means the ratio of the in the first counter and in the second counter records contained count value and that when the predetermined count value is reached a device responds to the second counter to determine the ' To operate drawing facility. 37. Apparatus according to claim 35, characterized in that that a second gate device, the second pulse device with the second counter and the the first gate device connects to the first counting device and that when the predetermined Count value responsive to the second counter means to the. second gate device to lock. 38. Apparatus according to claim 37, characterized in that a device is the ratio of the in the first and second counting means records counts contained therein, and that means is responsive to the recording means for the second gate means switch through and reset the first and second counters. ../54 209821/0884 39. Apparatus for determining the percentage of selected leukocytes in whole blood samples, characterized in that a device supplies the successive blood samples in the form of a continuous stream, that a device divides the continuous stream into several partial streams, each containing bursts of each of the successive blood samples , ψ Äass devices treat each of the substreams in order to detectably change the properties of selected leukocytes in the substreams, that a first device, based on the changed properties, discriminates and counts selected leukocytes in each of the batches contained in the substreams in order to generate a first batch for each sample batch To form count value that a second device detects leukocytes with changed and unchanged properties in each of the sample batches contained in the substreams, and counts in order to form a further counter value for each sample batch, the ratio tnis from the first and second count values corresponding to a sample batch in the partial flows indicates the percentage of selected leukocytes in the original blood sample, and that a device correlates the percentage proportions of the selected leukocytes in the assigned sample batches of the same original blood sample. 40. Apparatus according to claim 39, characterized in that a device separates the sample batches of each of the successive blood samples in the partial flows by an inert fluid stroke. ../55 209821/0884 -.55 - 41. Apparatus according to claim 39, d a;: characterized in that the treatment device contains a device which the properties of the leukocytes in a selected group of leukocytes in at least one of the partial flows changes demonstrably. 42. Apparatus according to claim 41, characterized in that that the first device individual leukocytes in the selected group in the one substream due to the discriminates changed properties and counts to form a third count ", the ratio the percentage value from the third count value and the count value formed by the second device Indicates the percentage of the selected group of leukocytes in the original blood sample. 43. Device according to claim 42, characterized in that that particular leukocytes in the selected group are treated in a different substream and that means the percentage of particular leukocytes from the percentage of selected ones Group of leukocytes subtracted to the percentage of remaining leukocytes in the selected Specify the group of the original blood sample. ../56 209821/0884 44. Apparatus according to claim 39 , characterized in that the second device assigned to at least one of the partial flows contains a device which discriminates and counts particular leukocytes in the one partial flow on the basis of unchanged properties of these particular leukocytes. 45. Device according to claim 43, characterized in that that the treatment means the spectral properties of the leukocytes in the selected group changes in at least one of the substreams, while the remaining leukocytes in this substream practically remain unchanged that a device this partial flow through a viewing chamber with a free passage of light directs that a device sends light through the light transmission path that the first Device is responsive to absorption of the light beam by a colored device responsive to a Scattering of light by colored and unstained leukocytes responds, with light scattering being a direct puncture of the cell size of a leukocyte is, and that the third device on the second device responds to a particular unstained type of leukocyte due to the size of the scattered Discriminate and identify light signal. ../57 209821/0884 46. Apparatus according to claim 45 »characterized in that. that another device responds to the third device for the particular unstained leukocytes to count, the ratio of the assigned count values of the assigned to a partial stream third and second establish the percentage of particular unstained leukocytes in the original Blood sample indicating. 47. Apparatus according to claim 46, characterized in that that a gate means the light scattering responsive second means and the third means connects to count the particular unstained leukocytes and that the gate means during the Occurrence of a colored leukocyte is blocked by the first device. Re / Li / Gr. 209821/0884 5S Blank page