WO1991012516A1

WO1991012516A1 - Process and device for determining clinical chemistry parameters inter alia

Info

Publication number: WO1991012516A1
Application number: PCT/DE1991/000086
Authority: WO
Inventors: Jörg Spindler
Original assignee: Spindler Joerg
Priority date: 1990-02-07
Filing date: 1991-01-30
Publication date: 1991-08-22
Also published as: DE4003604A1

Abstract

Clinical chemistry parameters, in particular the concentration of certain substances such as the enzyme GPT, the heparin cofactor AT3 and the blood clotting time factors FVIII and FIX, are determined by measuring the variation of optical density with time using a reader which measures the variation with time of the optical extinction coefficients of ninety-six samples on a microtitration plate. During the measurement, the microtitration plate is held stationary in the reader. Measurement is carried out by transilluminating the samples in the absence of diffuse light. The samples are previously conditioned and maintained with high precision at a constant measurement temperature.

Description

Device and method for determining clinical-chemical parameters and. a.

The invention relates to a device and a method for determining clinical-chemical parameters on the basis of the temporal change in the optical density of a larger number, in particular ninety-six, of liquid samples on a sample carrier, in particular for determining specific substance concentrations, for example the enzyme GPT, of the Heparin cofactor AT3 and the blood coagulation factors FVIII and FIX, for carrying out ELISA tests, agglutination tests, for example for determining blood groups and the like. a.

The concentration of glutamate pyruvate transaminase (GPT), also known as alanine aminotransferase (ALT), in the blood serum is significant for liver diseases, in particular NON-A-NON-B hepatitis, liver damage due to alcohol abuse and toxic liver damage for example through medication. The GPT determination is of great practical importance in the examination of the safety of preserved blood. The problem arises here of carrying out a rapid GPT determination on a large number of samples, emphasis being placed on a largely automatic sequence and positive sample identification, ie. H. a safe, non-manipulable assignment of the examined sample and the blood sample from which it comes.

The concentration of the blood coagulation factors FVIII and FIX is significant for a diagnosis of hemophilia. If there is a blood disorder, there is a deficiency of FVIII or FIX in the blood serum. The greater this deficit, the more acute the disease and the higher the demands on the treating doctor to find an optimal therapy. An exact determination of the concentration FVIII or FIX in the patient's blood serum is a necessary prerequisite for this.

The concentration of "heparin" cofactor AT3 in the blood serum is of crucial importance for the success of heparin therapy to prevent thrombosis, for example after cardiac surgery. Heparin only develops its anticoagulant function in the presence of "heparin" cofactor AT3. For the most accurate determination of the AT3 content in the patient's blood serum, a large number of samples must be examined in a short time under exactly defined, identical test conditions without external interference.

When determining the blood group, the hereditary antigen properties of the red blood cells are examined. In reaction tests with sera or reagents with known antibody properties, it is checked which antibody is used to induce red blood cell agglutination.

ELISA tests (Enzyme-Linked Immuno Sorbent Assay), ie heterogeneous enzyme immunoassays for the determination of immunogens and antibodies, as well as agglutination tests for the determination of blood groups are carried out, according to the applicant's knowledge, on blood sera with simultaneous examination of a large number of samples. A microtiter plate made of transparent material with, for example, ninety-six depressions for the samples serves as the sample carrier. The change in the optical density of the samples is recorded by the end point method with an automatic apparatus known as a "reader" in a simultaneous measurement of all samples, for which purpose light beams are sent through one or more samples at the same time and from the ratio of emitted to transmitted light intensity the extinction coefficient is determined. Reagents that change their optical extinction properties depending on a specific substance concentration in the blood serum, in particular the concentration of GPT, AT3, or FVIII. FIX, change, are known in the art. There are also extremely complex clinical-chemical large analyzers, large analyzers for blood group determination and large analyzers for coagulation, in which samples are treated serially, which results in very long measuring and processing times.

The object of the invention is to provide a device and a method which are not expensive to use and which allow rapid simultaneous measurement of clinical-chemical parameters, in particular the GPT or AT3 or FVIII or FIX concentration, and ELISA Tests and agglutination tests can be carried out on a large number, in particular ninety-six, of liquid samples, with positive sample identification.

This object is achieved by the device according to claim 1, the methods according to claims 17, 19, 21 and 23 and the use of a reader according to claim 27. Advantageous further developments of the invention are characterized in the dependent claims.

The device according to the invention uses a reader known from the prior art for simultaneous measurement of the change in the optical extinction coefficient of a large number, in particular ninety-six, of blood serum samples on a carrier. However, not every reader is suitable for the GPT or AT3 or FVIII or FIX determination or agglutination examination, since the change in the optical extinction coefficient to be demonstrated in a kinetic measurement is very small compared to ELISA tests. With the usual- The readers used are passed through a microtiter plate serving as a sample carrier under a row of parallel light beams which simultaneously irradiate several blood serum samples. The transport movement of the microtiter plate leads to sloshing of the blood serum samples, ie to dynamic changes in the level of their liquid level and corresponding to their optical densities, which massively falsify the measurement of small changes in the optical extinction coefficients. In addition, there are inaccuracies in the position of the light beams that do not exactly hit the sample center at the time of measurement. The simultaneous irradiation of several blood serum samples brings with it scattered light problems. The measurement of the intensity ratio of the emitted to transmitted light on a blood serum sample is falsified by scattered light which comes from other blood serum samples which have been irradiated at the same time, so that small changes in the optical extinction coefficient are difficult to detect.

To avoid these problems, according to the invention a reader designed for low-scatter measurement is used, in which the sample carrier is stationary during the measurement. Due to the latter fact, the liquid level of the samples is at rest during the measurement and there are no changes in the layer thickness due to liquid movements. In addition, the stationary arrangement of the sample holder is advantageous for maintaining the central measuring position of the light beams and for ensuring a uniform, constant measuring temperature, the importance of which is still to be discussed. It is not necessary to keep the transport path of a moving sample carrier, ie an area approximately twice the size of the sample carrier, at a uniform, constant temperature, but only the sample carrier itself. A movement promoted by the movement of the sample carrier Evaporation of liquid and the associated cooling effect are avoided.

A low-stray light measurement is ensured in the reader used in that the samples are irradiated individually. In addition, an aperture optic ensures low-scatter measurement. Even small changes in the optical extinction coefficient are detected very precisely in this way.

Some concentration determinations, for example those of GPT, have to be carried out according to international standards at very specific temperatures. The measured drop in the extinction coefficient over time is strongly influenced by temperature fluctuations, so that a constant and constant measuring temperature must be ensured over the surface of the sample carrier. Readers used for ELISA tests and agglutination tests are provided with a temperature-controlled heating unit which alone is not suitable for ensuring the uniformity and constancy of the measuring temperature required for a GPT determination.

In the device according to the invention it is therefore provided that the reader is located in a climatic chamber provided with a heating and / or cooling device, in which the measuring temperature of the samples can be set constantly with greater accuracy. The same or a second climatic chamber holds sample carriers for preheating the samples to a well-defined, uniform temperature.

According to the invention, the invention relates to a device for determining clinical-chemical parameters on the basis of the temporal change in the optical density of a larger number, in particular ninety-six, of liquid samples on a sample carrier, in particular for determining certain substance concentrations, for example the enzyme GPT, of the Heparin Cofactor AT3 and the blood coagulation factors FVIII and FIX, for carrying out ELISA tests, agglutination tests, for example for blood group determination, inter alia with one or more climatic chamber (s) provided with a heating and / or cooling device for receiving at least one sample carrier for one Pre-tempering of the samples and, if necessary, the use of reagents, as well as recording an apparatus (reader) designed for low-scatter measurement for the determination of the optical extinction coefficient of the samples with a measuring chamber in which a sample holder is recorded during the measurement and the temperature of the samples by the Heating and / or cooling device is constantly adjustable with high accuracy.

When using a reader which contains a temperature-controlled heating unit, by means of which the temperature of the samples in the measuring chamber can be influenced, the invention provides for the reader to be accommodated in a heatable and / or coolable climate chamber, whose internal temperature can be regulated to a constant, slightly lower than the measuring temperature of the samples.

In such a climatic chamber, the temperature-controlled heating unit of the reader achieves a measurement temperature which is uniform and constant over the surface of the sample carrier, as is required, for example, for GPT determination.

A temperature sensor can be arranged above the reader above its measuring chamber to regulate the internal temperature of the climatic chamber.

The climatic chamber receiving the reader can be a box accessible from above, in which the reader is so low that an air cushion of some height is located above the reader located. The air cushion contributes to a good temperature constant in the measuring range of the reader, since the air exchange with the surroundings takes place only slowly when the climatic chamber is opened.

The climatic chamber accommodating the reader can also be accessible from the side. It is recommended to use a lid or sliding door, e.g. B. double-wing sliding door, closable side opening in the chamber wall to install a curtain-like lock. When the reader is loaded with a sample holder, it is opened only as far as necessary, so that there is minimal air exchange with the surroundings and good temperature stability is ensured.

For preheating the samples and reagents, the device according to the invention advantageously has a preheating station with at least one support for at least one sample carrier, a channel running in the support or in its vicinity with a meandering path through which a heatable and coolable heat transfer medium extends can promote according to the countercurrent principle. An essentially horizontally laid heating-cooling coil through which the heat transfer medium flows can be considered as a support. Between the heating-cooling coil and the sample carrier, a grate that enables air circulation can be provided, which contributes to an even temperature distribution. However, a support plate that has one or more, preferably numerous, passage channels for the heat transfer medium can also be considered as a support. The passage channels can be meandered into the body of the support plate, which is preferably made of metal, for example aluminum, and can be sealed with a cover.

To regulate the preheating temperature, the invention provides a temperature sensor which is integrated into a temperature Reference liquid sample, for example a microtiter plate, is immersed, which, like other sample carriers, is located on a support of the pre-tempering station. The pre-tempering temperature can thus be set with very high accuracy.

The climatic chamber receiving the pre-tempering station is preferably accessible from the side. It is recommended to use a lid or sliding door, e.g. B. double-wing sliding door, closable side opening in the chamber wall to attach a curtain-like lock that is just opened as far as necessary when loading and removing sample carriers. The air exchange with the environment is minimal and a good temperature constant is guaranteed.

The curtain-like barrier preferably consists of flexible slats suspended from above, which laterally overlap one another and are preferably weighted on their lower edge.

A microtiter plate made of transparent material with ninety-six depressions, which preferably have a flat bottom, is preferably used as the sample carrier in order to ensure good optical properties.

According to the invention, a multichannel pipette, in particular ^" a ninety-six-channel pipette, is provided for, in particular, simultaneous application of samples to a sample carrier.

The pre-temperature control station and the reader can be accommodated in separate climatic chambers, preferably arranged one above the other and possibly connected by an elevator-like transport mechanism, while the multichannel pipette is preferably not necessarily next to it in a climatic chamber. This makes the pipetting process easier. The separate climatic chambers make it possible to set the preheating temperature of the samples and reagents slightly higher than the measuring temperature in the reader. A slight drop in temperature then occurs during the pipetting process the measuring temperature. The above-described arrangement of the climatic chambers is particularly suitable for a fully automatic machine. Note, however, that the climatic chamber for the pre-temperature control station, the multi-channel pipette and the climatic chamber for the reader can also be arranged next to one another.

However, there is also the possibility of accommodating a pre-tempering station, a pipetting station with a multi-channel pipette, in particular a ninety-six-channel pipette, and a reader not necessarily having its own heating unit in one and the same climatic chamber and preferably connecting them by a conveyor mechanism for sample carriers. This structure is preferred for a largely automatic measurement sequence.

In a first method variant for the determination of clinical chemical parameters, in particular for the enzyme diagnosis of blood serum, for example for the determination of glutamate pyruvate transaminase (GPT), the invention provides for a larger number, in particular ninety-six samples of an enzyme-specific fish (GPT -specific), with the enzyme (GPT) a detection reagent that promptly starts to erect, to be placed on a sample holder, the samples preheated at a well-defined temperature, the samples simultaneously treated with blood serum preheated at the same temperature, preferably shaken for some time , so that mixing takes place and well-defined liquid menisci and thus layer thicknesses are formed and in an apparatus designed for low-scatter measurement, in which the sample holder is accommodated in a stationary manner, a measurement of the temporal change in the optical with a high accuracy constant measurement temperature Extinct ionic coefficients of all samples. The blood serum samples are preferably prepared on a second sample holder, preheated together with the detection reagent samples and applied to the first sample holder with a multi-channel pipette, in particular a ninety-six-channel pipette.

The invention provides an alternative method variant propose to prepare a larger number, in particular ninety-six samples of an enzyme-specific (GPT-specific) with the enzyme (GPT) a detection reagent to be triggered by a start reagent mixed with serum on a sample holder, to pre-heat the samples at a well-defined temperature, the samples to be mixed simultaneously with the starting reagent preheated at the same temperature, preferably to be shaken for some time, and to carry out the measurement in the reader. The starting reagent, too, is preferably preheated together with the samples and applied to the sample carrier with a multiarial pipette, in particular a ninety-six-channel pipette.

In a third variant of the method, the invention provides for a larger number, in particular ninety-six, of blood serum samples to be placed on a sample carrier, for the samples to be preheated at a well-defined temperature, for the samples to be simulated with an enzyme-specific (GPT-specific) preheated at the same temperature, with the enzyme (GPT) to add a reaction reagent which shows a prompt reaction, preferably to shake for some time, and to carry out the measurement in the reader. Here too, the detection reagent is preferably preheated together with the samples and applied to the sample carrier with a multi-channel pipette, in particular a ninety-six-channel pipette.

In a fourth method variant for determining clinico-chemical parameters, in particular for examining blood coagulation behavior, for example for determining blood coagulation inhibitors, preferably heparin cofactor AT3, the invention provides for a larger number, in particular 96 samples of a factor-specific (AT3 -specific) detection reagent (o <-thrombin), which shows the factor promptly starting a reaction, on a sample holder, preheating the samples at a well-defined temperature, pretempering the samples simultaneously with the same temperature. - 11 -

the diluted blood serum, preferably shaking for some time, and incubating at a constant temperature with high accuracy for some time, preferably about 3 minutes, and then with a chroogenic substrate (HD-CHA-But -Arg-pNA) and to carry out a measurement of the temporal change in the optical extinction coefficient of all samples in an apparatus designed for low-scatter measurement, in which the sample carrier is accommodated in a stationary manner, with a high accuracy constant measuring temperature. The detection reagent and the chromogenic substrate are preferably preheated together with the serum samples and applied with a multichannel pipette, in particular a 96-channel pipette, to the sample carrier serving as the measuring plate.

With all process variants, it is advisable to carry out the shaking and incubation in the apparatus (reader). You can advantageously use a vibrating mechanism and the time control of the reader.

Attention should be drawn to the possibility of covering the samples during the pre-incubation, e.g. B. by means of a film web, a transparent cover plate or the like, and optionally also carry out the optical measurement in the reader through the cover plate. In this way, the evaporation of liquid and the associated evaporative cooling are inhibited and, if necessary, the optical components and the electronics of the reader are protected.

Finally, the invention relates to the use of an apparatus (reader) designed for low-scatter measurement for measuring the optical extinction coefficient of a larger number, in particular ninety-six liquid samples on a sample carrier which is stationary during the measurement, for determining clinical-chemical parameters on the basis of the change over time optical density, in particular for determining certain substance concentrations, for example the enzyme GPT, the heparin cofactor AT3 and the blood coagulation factors FVIII and FIX, for carrying out ELISA tests, agglutination tests, for example for determining blood groups, among other things, the samples placed on the sample carrier being preheated and brought to a well-defined temperature and during the measurement in the reader for determining the change over time optical extinction coefficients of the samples are kept at a measuring temperature constant with high accuracy.

The invention is explained in more detail below with the aid of examples.

example 1

In the course of an examination of the safety of preserved blood, ELISA tests for CMV, HCV, HIV I and II antibodies and HBS antigen, heme agglutination tests for antibody TPHA and a GPT determination are to be carried out. For this purpose, in a first pipetting run, groups of up to ninety-six blood preserve tubes each are taken with a pipetting robot and placed on five microtiter plates, one of which serves as the primary plate for the GPT determination. After appropriate treatment, the other micro-titer plates are used in the same system for the ELISA tests and heme agglutinatin tests. For a blood group determination, further serum samples are taken in a second pipetting run with the pipetting robot and used for an agglutination examination in the same system.

The primary plate for the GPT determination is a flat-bottom plate with ninety-six depressions, which have a flat bottom and each take a serum sample. The assignment of the sample position on the primary plate and the blood tube, and thus the blood sample from which the sample comes, is unambiguous, and it is automatically determined during sampling. logged.

A GPT-specific detection reagent is prepared on a further microtiter plate, which serves as a measuring plate below. The measuring plate is made of transparent material. It is a flat floor slab, the depressions of which have a flat floor. The primary plate is also a flat-bottom plate of this type, which is important for preheating to the same temperature. The detection reagent shows a prompt reaction with GPT, during which it changes its optical extinction properties depending on the GPT concentration in the sample.

The primary plate with the blood serum samples and the measuring plate with the detection reagent are covered, brought together into a preheating station and left there until the blood serum and detection reagent have reached a desired preheating temperature. The level of the preheating temperature depends on the measuring temperature at which the GPT determination is to be carried out. A measuring temperature of 25 ° C, 30 ° C or 37 ° C corresponds to international standards. The preheating temperature is preferably a few tenths of a degree higher than the measuring temperature. If the latter is 25 "C, a preheating temperature of 25.2 ° C is recommended. Blood serum samples and detection reagent are evenly heated to this temperature.

The primary plate and the measuring plate are then removed from the pre-tempering station. The blood serum samples are pipetted from the primary plate onto the measuring plate with a ninety-six-channel pipette, with all detection reagent samples being mixed with the blood serum at the same time. The measuring plate maintains a temperature around the desired measuring temperature for the required time solely on the basis of its heat capacity. The measuring plate is then covered with a transparent cover and placed in a Thermomax reader from Molecular Division Inc., which automatically measures the temporal drop in the optical extinction capacity of the samples. The reader contains a jogger, which is used to shake the plate for some time in a pre-incubation phase. In addition, this type of reader is characterized in that the microtiter plate is stationary during the measurement and that the samples are illuminated individually at intervals in order to determine the optical extinction coefficient from the ratio of emitted and transmitted light intensity. Stare light is effectively suppressed by an aperture optic. Obviously, the invention is ^' not restricted to a specific type of reader, provided that the movements of the liquid, which distort the measurement result, are avoided only by a stationary arrangement of the microtiter plate and effective stray light suppression takes place.

The reader is in a climate chamber in the form of a thermally insulated box with a lid on the top. There is a distance of at least 10 cm between the cover opening and the reader, so that an air cushion which slows down the heat exchange can build up above the reader. A pipe coil is laid on the inside walls of the box, through which water circulates in a closed circuit, which is tempered with a commercially available heating / cooling unit. The ambient temperature of the reader is measured with a temperature sensor which is located above the reader above its measuring chamber. The water temperature in the heating / cooling circuit is controlled so that the ambient temperature of the reader remains constant and is approx. 5 ° C below the measuring temperature at which the GPT determination takes place. At a measuring temperature of 25 ° C, the ambient temperature of the reader is approx. 20 ° C. It should also be noted that the reader can also be accommodated in a refrigerator-like climate chamber, which can have a loading and removal opening that can be closed at the side with sliding doors.

The reader has a heating unit and an internal temperature control with a sensor which detects the air temperature in the measuring chamber in order to regulate it to the desired measuring temperature. Because the ambient temperature of the reader is kept constantly slightly below the measuring temperature, the internal temperature control of the reader achieves the exact observance of the measuring temperature of, for example, 25 ° C. which is necessary for the GPT determination. Structural changes to the reader are not necessary. In the context of the invention, of course, there is also the possibility of a reader in a different way with a heating-cooling unit z. B. equip according to the countercurrent principle for precise control of the measuring temperature.

After inserting the measuring plate into the measuring chamber of the reader, a first absolute measurement of the optical extinction coefficient of the samples is carried out. The pre-incubation phase then follows, in which the measuring plate is shaken mechanically in order to achieve a thorough mixing of the detection reagent and blood serum and to form well-defined liquid menisci and thus layer thicknesses. You then wait until the samples have calmed down and begin measuring the reaction kinetics, i.e. in the pre-incubation phase. H. of the time drop E / f_ t of the optical extinction coefficient in the ninety-six samples. At the beginning of the actual measurement phase after the pre-incubation phase, a second absolute measurement of the optical extinction coefficient is carried out, which is also included in the ΔE / A t measurement. The GPT concentration can be determined from the measurement result Λ E / Δt.

For the separation of highly pathological samples with very high E / Δt, ie high substrate consumption, the result of the absolute measurement at the beginning of the pre-incubation phase and at the beginning of the actual measurement phase is used. The measured values must not fall below a set threshold. Furthermore, the result of the Δ E / t measurement in the pre-incubation phase is compared with that of the Δ E / Λt measurement in the actual measurement phase in order to identify incorrectly low measured values in the low GPT concentration range or very high substrate consumption =

Nonlinearities in the time course of ΛE / Δt indicate disturbance kinetics and beginning reagent consumption. Disturbance kinetics can also be identified using an atypical correlation coefficient (r value) of the measured values. Strongly lipemic or hemolytic samples can be identified on the one hand by an atypical r value, and on the other hand on the basis of the absolute value of the optical density obtained at the beginning of the measurement phase, which should not exceed a predetermined threshold value. The simultaneous occurrence of an atypical correlation coefficient and an atypical ratio of ΔE / Λt in the pre-incubation and measurement phase indicates a malfunction of the multichannel pipette, a faulty blood sample or the like.

In parallel to the described ninety-six-channel GPT determination, ELISA tests and agglutination tests can be prepared in a known manner by the same laboratory worker and then carried out in the same system.

Example 2

In contrast to Example 1, the primary plate with the blood serum samples also serves as a measuring plate. A detection reagent is pipetted onto the primary plate with a ninety-six-channel pipette, which reagent is changed with GPT with a change in the optical absorbance. ability to react. However, the reaction is only triggered by adding a start reagent. Detection reagent and blood serum are mixed by gently shaking the microtiter plate. The microtiter plate is brought into the preheating station and preheated in the manner described together with a dish containing the starting reagent. The bowl is designed so that the start reagent can be removed with the ninety-six-channel pipette. When the desired preheating temperature has been reached, the microtiter plate and the tray are removed from the preheating station, the starting reagent is applied to the microtiter plate with the ninety-six-channel pipette, and the latter is placed in the reader to measure the reaction kinetics.

Example 3

As in Example 2, the briar plate with the blood serum samples also serves as a measuring plate for the reader measurement. The primary plate is preheated together with a dish of detection reagent which, with GPT as in Example 1, shows a prompt reaction with a change in the optical extinction capacity. After the preheating has been carried out, a reagent is added from the dish onto the microtiter plate using a ninety-six pipette and the reader measurement is carried out.

Example 4

After cardiac surgery, the patient is given heparin to prevent thrombosis. Since heparin develops its anticoagulant function only in the presence of the so-called "heparin" cofactor AT3, the content of AT3 in the patient's blood serum should be determined.

For this purpose, serum samples on a microtiter plate (primary plate) in a ratio of approx. 1:20 to 1:40 with physiological diluted saline solution and pre-tempered together with a second microtiter plate serving as a measuring plate, on which a detection reagent {X-thrombin) is attached, and a dish with a chromogenic substrate (HD-CHA-Bύt-Arg-pNA). The bowl is designed so that chromogenic substrate can be removed from it with a ninety-six-channel pipette. When the desired pre-tempering temperature has been reached, the microtiter plates are removed from the pre-tempering station and pipetted serum samples to the detection reagent using a ninety-six-channel pipette. To determine the thrombin enzyme blank value, a corresponding amount of physiological saline solution was added to a few depressions in the primary plate instead of blood serum. The measuring plate is then placed in the apparatus (reader) and shaken and incubated for some time, then, with the aid of a sixty-six-channel pipette, mixed with the chromogenic substrate at exactly the same temperature and placed back in the reader to measure the change in the optical extinction coefficients over time.

In the reaction that follows, a portion of the detection reagent from AT3 molecules contained in the serum is captured and the remaining detection reagent is detected by a color reaction with the chromogenic substrate, which undergoes a color change from transparent to colored. The increase in the optical extinction coefficient E / Λt over time is measured in the reader. For the evaluation, the difference between the serum values and the thrombin enzyme blank values is formed, which is directly proportional to the AT3 content.

drawing

The structure of the pre-tempering station is described below with reference to the drawing. Show it: Fig. 1 is a side view of a support for one in the

Pre-tempering station - microtiter plate;

Figure 2 is a plan view of the support with a view in the direction II of Fig. 1.

3 shows the front view of a climatic chamber provided with a hinged lid, in which the support is located, with the lid closed;

FIG. 4 shows a front view of the climate chamber corresponding to FIG. 3 with the cover open; and

5 shows a side view of the climatic chamber when a microtiter plate is removed with a view in direction V of FIG. 4.

The support essentially consists of a heating / cooling coil 10, which is laid horizontally in a meandering path and through which a heat transfer medium, preferably water, flows according to the countercurrent principle. The forward and return paths of the heating-cooling coil 10 each lie parallel to one another and together form a rectangular spiral of constant slope. On the heating-cooling coil 10 there is a grate 12 on which a number of microtiter plates 14 are placed. The grate 12 enables air circulation that is useful for rapid temperature compensation.

In an alternative embodiment, the support is a support plate which is provided with passage channels for the heat transfer medium according to the countercurrent principle (not shown).

One of the microtiter plates 14 on the support contains a temperature reference liquid sample which is not required for the measurement of clinical-chemical parameters. A blood serum sample, detection reagent sample, starting reagent sample or other liquid sample can be considered as the temperature reference liquid sample. A temperature sensor 16, which is located in an external temperature control circuit and is used to regulate the preheating temperature, is immersed in the liquid sample. serves the samples on the edition. If the temperature measured with the temperature sensor 16 is below the desired pre-tempering temperature, the heat transfer medium flowing through the heating-cooling coil 10 is heated and, in the other case, cooled.

The support is located in a thermally insulated box 18, which is closed at the top with a hinged lid, not shown. The hinged lid is only opened for the first loading of the box 18 with microtiter plates and for maintenance and cleaning purposes. In contrast, the hinged lid remains closed during operation. For loading and removing microtiter plates 14 during operation, the box 18 is provided with a side opening which can be closed with a second hinged lid 20, a sliding door or the like. Behind the hinged lid 20 there is a curtain-like lock, which consists of flexible slats 22 suspended from above. The slats 22 are arranged next to one another with lateral overlap and weighted with weights 24 on their lower edge.

When loading and removing microtiter plates 14 through the lock, the lamellae 22 are deflected upward only in the required width and also only to the extent necessary, so that there is a minimal exchange of air with the surroundings and the temperature in the box 18 in remains essentially constant.

The device and method according to the invention are not restricted to the detailed descriptions of GPT, AT3, FVIII and FIX. Rather, other clinical chemical parameters and blood coagulation factors can also be determined in a similar manner, in particular aldolase, AP, alcohol, amylase, ANP, bilirubin, calcium, cholesterol, cholinesterase, CK, FE, Emit, total protein, glucose, GLDH, GOT , / -GT, uric acid, urea, 0 ^ -HBDH, creatinine, lactate, LDH, LAP, lipase, acidic phosphatase, parameters to be determined by turbidimetric methods, triglycerides, PT, APTT, TZ, fibrinogen, factors exogenous and endogenous system, Procomplex TM, hepatocomplex TM and others.

List of reference numbers

Heating-cooling coil grate microtiter plate temperature sensor climate chamber hinged cover slat weight

Claims

Expectations

1.Device for determining clinical-chemical parameters based on the temporal change in the optical density of a large number, in particular ninety-six, of liquid samples on a sample carrier, in particular for determining certain substance concentrations, for example the enzyme GPT, the heparin cofactor AT3 and the blood coagulation factors FVIII and FIX, for carrying out ELISA tests, agglutination tests, for example for blood group determination and the like. a. with one or more climatic chamber (s) provided with a heating and / or cooling device for receiving at least one sample holder for preheating the samples and, if necessary, for the use of reagents, and for receiving an apparatus (reader) designed for low-scatter measurement for determining the optical extinction coefficients of the samples with a measuring chamber in which the sample holder is received stationary during the measurement and the temperature of the samples can be adjusted with high accuracy by the heating and / or cooling device.

2. ^• Device according to claim 1, characterized in that the reader contains a temperature-controlled heating unit through which the temperature of the samples in the measuring chamber can be influenced, and that the reader is located in a heatable and / or coolable climatic chamber, the Internal temperature can be regulated to a constant, somewhat lower than the measuring temperature of the samples.

3. Apparatus according to claim 1 or 2, characterized gekennzeich¬ net that to regulate the internal temperature of the climatic chamber above the reader above its measuring chamber a temperature door sensor is arranged.

4. Device according to one of claims 1 to 3, characterized in that the climatic chamber is accessible from above, and that there is an air cushion of some height above the reader.

5. Device according to one of claims 1 to 3, characterized in that the climatic chamber is accessible from the side, it being possible for a curtain-like lock to be fitted in front of the opening in the chamber wall which can be closed by a cover or a sliding door.

6. Device according to one of claims 1 to 5, characterized in that it has a pre-tempering station serving for preheating the samples with at least one support for at least one sample carrier, with a channel running through or in the support or in the vicinity with a meandering path a heatable and coolable heat transfer medium can be pumped according to the countercurrent principle.

7. The device according to claim 6, characterized in that the support is a substantially horizontally laid, flowed through by the heat transfer medium heating-cooling coil (10).

8. The device according to claim 7, characterized in that between the heating-cooling coil (10) and the sample carrier an air circulation grating (12) is provided.

9. The device according to claim 6, characterized in that the support is a support plate, the one or has several, preferably numerous, passage channels for the heat transfer medium.

10. Device according to one of claims 1 to 9, characterized in that there is a temperature reference liquid sample, for example a microtiter plate, on which the temperature sensor (16) is used to regulate the pre-tempering temperature.

11. The device according to one of claims 1 to 10, characterized in that the climatic chamber (18), in which the pre-tempering station is located, is accessible from the side, and that in front of the closable with a cover (22) or a sliding door Opening in the chamber

• a curtain-like barrier is attached to the wall.

12. The apparatus of claim 5 or 11, characterized gekennzeich¬ net that the lock consists of suspended from above, flexible slats (24) which overlap each other laterally and can be weighted on its lower edge (26).

13. Device according to one of claims 1 to 12, characterized in that the sample carrier is a Mikrotiter¬ plate made of transparent material with preferably ninety-six depressions, which preferably have a flat bottom.

14. Device according to one of claims 1 to 13, characterized in that a multichannel pipette, in particular a ninety-six channel pipette, is provided for, in particular, simultaneous application of samples to a sample carrier.

15. The device according to one of claims 1 to 14, characterized characterized in that the pre-temperature control station and the reader are accommodated in separate climatic chambers, and that the multi-channel pipette, in particular ninety-six pipette, is located outside of it.

16. Device according to one of claims 1 to 14, characterized in that a pre-temperature control station, a pipetting station with a multi-channel pipette, in particular a ninety-six channel pipette, and a reader not necessarily having its own heating unit, are accommodated in one and the same climatic chamber and preferably by a conveyor mechanism for sample carriers are interconnected.

17. A method for determining clinical-chemical parameters, in particular for enzyme diagnosis in blood serum, for example for determining glutamate-pyruvate transaminase (GPT), in which a larger number, in particular ninety-six, samples of an enzyme-specific (GPT-speci - fish), with the enzyme (GPT), a detection reagent which shows a rapid onset of reaction is prepared on a sample carrier, the samples are preheated at a well-defined temperature, the samples are simultaneously treated with blood serum which has been preheated at a well-defined temperature, preferably shaken for some time and in one on a low-scatter measurement designed apparatus, in which the sample holder is accommodated stationary, with a high accuracy constant measurement temperature, a measurement of the temporal change in the optical extinction coefficient of all samples is carried out.

18. The method according to claim 17, characterized in that a larger number, in particular ninety-six, blood serum samples are prepared on a second sample carrier, the blood serum samples together with the detection reagent. Apply samples preheated and with a multi-channel pipette, in particular a ninety-six-channel pipette, to the first sample carrier.

19. A method for determining clinical-chemical parameters, in particular for enzyme diagnosis in blood serum, for example for determining glutamate pyruvate transaminase (GPT), in which a larger number, in particular ninety-six samples of an enzyme-specific (GPT-specific fish), uses the enzyme (GPT) to prepare a detection reagent to be triggered by a start reagent mixed with blood serum on a sample holder, the samples are pre-tempered at a well-defined temperature, the samples are simultaneously mixed with a start reagent pre-tempered at a well-defined temperature, preferably shaken for some time and in one on a low-scatter measurement device, in which the sample holder is accommodated stationary, a measurement of the temporal change in the optical extinction coefficient of all samples is carried out with high accuracy constant measuring temperature.

20. The method according to claim 19, characterized in that the starting reagent is pre-tempered together with the samples and applied to the sample carrier with a multi-channel pipette, in particular a ninety-six-channel pipette.

21. A method for determining clinical-chemical parameters, in particular for enzyme diagnosis on blood serum, for example for determining glutamate pyruvate transaminase (GPT), in which a larger number, in particular ninety-six, of blood serum samples are prepared on a sample carrier , the samples pre-tempered at a well-defined temperature, the samples simultaneously with a well-defined temperature enzyme-specific (GPT-specific) which has been preheated to the temperature and with which the enzyme (GPT) a detection reagent which shows a prompt reaction is added, preferably shaken for some time and in an apparatus designed for low-scatter measurement, in which the sample holder is held stationary is a measurement of the temporal change in the optical extinction coefficient of all samples is carried out at a constant temperature with high accuracy.

22. The method according to claim 21, characterized in that the detection reagent is preheated together with the blood serum samples and applied to the sample carrier with a multi-channel pipette, in particular a ninety-six channel pipette.

23. Method for determining clinical-chemical parameters, in particular for examining the blood coagulation behavior, for example for determining blood coagulation inhibitors, preferably heparin cofactor

AT3, in which a larger number, in particular 96 samples of a factor-specific (AT3-specific) detection reagent (C <-trombin) with the factor promptly reacting, is prepared on a sample carrier and the samples are preheated at a well-defined temperature, the samples are simultaneously mixed with diluted blood serum preheated at the same temperature, preferably shaken for a time and incubated at a constant temperature with high accuracy for a few, preferably 3 minutes and then with a chromogenic substrate (HD-CHA-But-Arg-pNA) preheated at the same temperature ) offset and in an apparatus designed for low-scatter measurement, in which the sample carrier is accommodated stationary, a measurement of the change in time of the optical extinction coefficients of all samples.

24. The method according to claim 23, characterized in that the detection reagent and the chromogenic substrate are preheated together with the serum samples and applied with a multi-channel pipette, in particular a 96-channel pipette, to the sample carrier serving as the measuring plate.

25. The method according to any one of claims 17 to 24, characterized in that the shaking and incubation in the apparatus (reader) is carried out.

26. The method according to any one of claims 17 to 25, characterized in that the sample carrier during the pre-incubation z. B. by means of a film web, a transparent cover plate or the like, and optionally also carries out the optical measurement in the apparatus (reader) through the cover plate.

27. Use of an apparatus (reader) designed for a low-scatter measurement measurement for measuring the optical extinction coefficient of a large number, in particular ninety-six, of liquid samples on a sample carrier, which is stationary during the measurement, for determining clinical-chemical parameters on the basis of the change over time the optical density, in particular for determining certain substance concentrations, for example the enzyme GPT, the heparin cofactor AT3 and the blood coagulation factors FVIII and FIX, for carrying out ELISA tests, agglutination tests, for example for determining blood groups and the like. a. , wherein the samples placed on the sample holder are preheated and brought to a well-defined temperature and are kept at a constant temperature with high accuracy during the measurement in the reader to determine the temporal change in the optical extinction coefficient of the samples.