CH637216A5 - Device for treating a measuring liquid for use in analytical optical devices - Google Patents

Description

637 216 2

PATENT CLAIMS especially for the analytical examination of body fluids

1. Device for preparing a measuring liquid used for keiten.

Use in analytical optical devices, consisting of a biochemical test in the area of hematological, clinical-chemical and sealed, with a precisely dosed reaction solution, increasingly offers "pre-filled cuvette and a capillary 5 tests" that allow the user without having to prepare the sample liquid into the reaction solution reagents quickly and easily.

solution, whereby the optical properties of the measuring liquids hematological, clinical-chemical and biochemical

Set the speed in a characteristic way, thereby carrying out familiar parameters.

indicates that a part of the reactants in the solid phase in these «ready tests», also mono-test, one-test or one-

The capillary (5) or a white glass test used for dosing are characterized by the fact that unstable capillaries (6) that can be inserted into the cuvette are used to house components of the ready-to-use reagent in mostly

is. freeze-dried form in glass or plastic containers

2. Device according to claim 1, characterized, be preserved.

that the reactants in finely divided form on the inside Before use, a solvent is applied to the capillaries (5,6). 15 re may contain substances required for the complete reagent,

3. Device according to claim 1-2, characterized gekennzeich- added. The reagent is then ready for use, so that the reac- For some determinations, e.g. In the case of the hemoglobin solution (1) in the cuvette from a phosphate-buffered atmosphere, the ready-to-use reagent can also be in the glass of potassium hexacyanoferrate (III) / sodium chloride solution or plastic containers can be present.

and the capillary (5) used for metering the sample. The usable reagent is then usually coated with a potassium cyanide. Pipette to ensure accurate dosing

4. Apparatus according to claims 1-2, characterized in that the sample (liquid, blood, plasma, serum) is analyzed in such a way that the reaction solution is used to determine bilirubin in the blood. The reagent-sample mixture is then made up of 0.1 N hydrochloric acid and next to it the measuring vessel, usually a glass or quartz cuvette, and the capillary (5) used for the blood sample, another capillary 25 in a measuring device, e.g. analyzed with a photometer.

(6) is envisaged with 2,5-dichlorophenyldiazonium chloride. More recently closable cuvettes (DT-OS-

is coated. 2 422 260), which is known as a plastic disposable measuring

5. Device according to claim 1-2, characterized gekennzeich- cuvettes (preferably made of polystyrene) and with net that the reaction solution are sealed airtight from a plastic lid for cholesterol determination.

a potassium phosphate-buffered phenol-methanol solution 30

with the addition of hydroxypolyethoxydodecane and besides welding, e.g. using ultrasound, connected to the cuvette,

the cap used for metering in the serum or plasma. The lid has a predetermined breaking point, which can be broken open by means of a pillare (5), another capillary (6) for removing a pin. Through the opening created in the

Cholesterol esterase, cholesterol oxidase and peroxidase best cuvette cover, the sample to be analyzed can be provided in the cuvette-containing enzyme mixture, which can be introduced with 4-aminophe-35.

is coated with nazon. The cuvette itself already contains the complete reagent

6. Device according to claims 1-2, characterized in liquid. For the photometric determination of the erythrocyte, that in order to carry out an enzymatic blood sugar count, e.g. the user has only 5 [xl blood with a mood according to Trinder, the reaction solution (1) consists of phosphate-volume-calibrated glass capillary through the opening in the dec-buffered phenol and in addition to the 40 kel dose into the cuvette. As soon as the blood has been completely mixed with a capillary (5) used with the serum or plasma sample, the photometric measurement of the other capillary (6) can be provided, which is carried out with a mixture of. This process is particularly characterized by the fact that glucose oxidase, peroxidase and 4-aminophenacone are coated so that the user neither prepares the reagent itself nor is it. still have to solve, that for the analysis auxiliary devices such as

7. The device according to claim 1-2, characterized marked 45 pipettes required. This considerably combines the analysis that an enzymatic blood glucose monitoring is carried out so that even non-specially trained people can perform the reac- tion correctly using the glucose dehydrogenase method.

tion solution from a phosphate buffer solution with a pH value The methods described have the following disadvantages:

7,6 exists and in addition to the capillary used for dosing - In the “ready tests”, pipetting lare (5) usually requires another capillary (6), which is carried out with one. This will be expensive and sensitive

Mixture of glucose dehydrogenase, mutarotase and auxiliary devices such as Eppendorf pipettes required. The pipette

Nicotinamide adenine dinucleotide (NAD) is coated. tion itself is a source of error. Often the required is listed

8. The device according to claim 1-2, characterized in solvent, e.g. distilled water, not in the required net, that is available to determine the cholinesterase activity by quality (e.g. quartz distilled water).

Ellman the reaction solution from a phosphate buffer solution with 55 The complete reagent or the complete reagent

a pH value of 7.2 and, in addition to the sample mixture for metering, must also be placed in a measuring device, e.g. a glass kitchen

capillary (5) used, a further capillary (6) is transferred before the actual measurement process is complete, which can take place with a mixture of acetylthiocholine iodide and. This is an additional step that

Dithiobis-2-nitrobenzoic acid is coated. includes other possible errors, e.g. not quite clean

60 bare or not completely dry (volume error!) Cells or

Detergent residues in the cuvettes.

Overall, however, the handling of these “ready tests” is so complicated that the invention relates to a device for processing, that one can justifiably not speak of a measuring liquid for use in analytical optical devices, and that it can be said for non-spewing and assumes a closed cuvette filled with a precisely 65 trained person and a whole series of error-dosed reaction solution and one that results in the sources when handling these tests.

Cuvette-insertable capillary for metering the samples - The test described in DT-OS-2 422 260 fulfills liquid in the reaction solution. In particular, the invention largely meets the requirements for a “ready test”.

3,637,216

The user does not have to dissolve reagents, mix the capillary used or another solution that is separated into the cuvette exactly, nor is the finished reagent-insertable capillary accommodated.

Transfer the sample mixture into a measuring vessel. It simply has to be. The reaction partners are preferably in the form of a finely divided sample which is to be analyzed and introduced into the cuvette in precisely metered form on the inner wall of the capillary.

gen, this can e.g. simply by volume-calibrated glass 5 In this way, the spectrum of the reactants pillaren can take place and the cuvette can be expanded considerably into the photometer. Often, all re can be managed. Action partners are not kept together in a solution.

This eliminates possible sources of error (pipetting), since they then become unstable within a short time or are switched off beforehand and the analysis process thus reacts with one another. By relocating such unstable compartments that even untrained personnel can correctly analyze the components in the capillaries, this problem can be carried out in elegan. ter solved. This allows further reactions for the in

Unfortunately, however, there are only a few reagents for biochemical, hematological and DT-OS-2 422 260 analytical methods described in clinical-chemical analysis which are closed in the past and which have been used for a long time due to the instability of the ready-to-use state ( 6 months) shelf life were not suitable for this. Those with a reak are (sales!). 15 coated capillaries remain dry-treated

This is especially true for the modern enzymatic measurement, stable over a long time (more than 6 months), while the methods. So e.g. for the determination of blood sugar with glucose-stable reaction partners in the solution in the usual way, ge-oxidase / peroxidase, dosed with hexokinase / glucose-6-phosphate-De-nau, welded into the measuring cell, hydrogenase or with glucose dehydrogenase / mutarotase, the user can now also take the sample (liquid, blood, plasma, serum) for the enzymatic determination of uric acid, the 20 a) with the capillary coated on the inner surface of the capillary urea, the triglycerides and cholesterol and also for the activity determination of the enzymes Insert the filled capillary into the single-use plastic cuvette.

But also reagents for non-enzymatic determinations. With a very simple and quickly executable gene, the reagent is often not ready for use over a long period of time (6 months) and the reagent is stable to analyze. This applies e.g. exactly dosed with 25 de sample for the determination of bilirubin. After a possibly necessary re-diazotized sulfanilic acid according to Jendrassik and Gróf [2] or with an action time, the sample can be analyzed in a measuring device (e.g. Photome-2,5-dichlorophenyldiazonium salt according to Wahlefeld et al. [3], ter).

likewise for the determination of creatinine with picric acid / In this case the capillary used must of course

NaOH according to Popper et al. [4]. be volume calibrated and that on the inside surface of the

The use of disposable measuring cuvettes brings an additional 30 pillare applied component of the reagent with the filling problems. Glass does not falsify as a material for disposable measuring volumes.

cuvettes, since glass cuvettes in the capillary coated on the inner surface for the photometric measurement are much too expensive in the quality required for the solutions. Insert the prefabricated plastic disposable measuring cell and thereby complete the reagent. The pro-the use of plastics to be analyzed, e.g. Polystyrene, allowed 35 be (liquid, blood, plasma, serum) can then be added - inexpensive mass production of disposable measurements (e.g. with a volume-calibrated capillary), cuvettes, but often brings problems with regard to the hold-up after one necessary reaction time, the sample availability of the reagents. be analyzed in a measuring device (e.g. photometer). Got to

So polystyrene is not absolutely impermeable to water vapor. a blank reagent value can be determined during the determination. If, on the other hand, no suitable measures are taken, this can be changed so that the volume of the reagent liquid in the verb can be determined before adding the analyte to be analyzed.

closed cuvette during storage. Since a sample blank value must be determined, the analyzes to be carried out are always based on a known, and expediently, the sample first arrives in the pre-prepared art-defined mixing ratio, so you get a disposable measuring cell, determine the blank value, wrong results. 45 the reagent is pletted by adding the coated capillary

Polystyrene is also permeable to many gases. This has an effect and then determines the analytical value. This can e.g. negative on the shelf life of the reagent for determination in a plastic disposable measuring cuvette both of the samples - from hemoglobin as hemoglobin cyanide. This reagent blank as well as the analytical value can be determined. As a result, in addition to a buffer substance and a detergent to save, a cuvette and the amount of sample that are otherwise required for molyzing the erythrocytes also contain potassium hexacyanoferrate (III), which would make it necessary to determine the sample blank value. Hemoglobin is oxidized to hemiglobin and KCN converts the hemiglobin c) with the capillary coated on the inner surface into hemiglobin cyanide. The hemiglobin cyanide becomes another unstable component, e.g. an enzyme suspension is then determined photometrically. or take up the suspension of an enzyme mixture into which

The KCN contained in the reagent and HCN are in the prefabricated plastic disposable measuring cell and the gas space above the liquid is in equilibrium. HCN can thereby complete the reagent.

diffuse through the polystyrene wall of the cuvette, so the addition of the sample can depend on whether a reagent

that the CN ~ content of the reagent with a half-life of empty value or a sample empty value has to be determined decreases about 4 weeks ago. This results in a - for or after the addition of the unstable components.

Sale - insufficient shelf life of the reagent. The method described under a) has e.g. especially

The object of the present invention was now to overcome the 60 and cyanide proven in the determination of hemoglobin as the hemoglobin life-time problems described above.

To develop ready-made tests in which pre-made plastic 1.25 ml of the reagent complete with the exception of the KCN

Disposable cuvettes can be used and which allow the zes to be inserted into the plastic disposable measuring cuvette without additional devices such as e.g. Pipettes and welds. The KCN (70 [xg per capillary) is finely distributed without any special training in order to obtain correct analyzer 65 on the inner surface of a volume-calibrated result. Capillary of 5 [il content. With this capillary the sample

This object is achieved according to the invention in that blood is taken up in this case. Due to the capillary action, some of the reactants in the solid phase in which the capillary is to be dosed in will fill up automatically as soon as it is filled with the blood

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touches. The KCN dissolves immediately in the blood and - The use of plastic disposable measuring cuvettes does not measurably affect the accuracy of the sample dosage. allows the same vessel for sample preparation and for

The capillary will be used in the measurement after it has filled with blood. As a result, the user needs the

Plastic disposable measuring cell inserted. Due to a light test, no longer in a special reaction vessel with the

Shake the measuring cuvette, mix the capillary content with the cu 5 reagent and then mix it into the measuring vessel into the vette content, thereby transferring the reagent.

completed and the sample dosed. After a response time this means both a cost saving (no reac-

of 3 minutes, the hemoglobin content of the sample may be necessary directly in the test vessels!) as well as a work simplification.

be determined with a photometer. - Since the prefabricated plastic disposable measuring cell is all

The method described under b) has been precisely metered, especially in the case of liquid components of the reaction solution, contains the bilirubin determination with 3,5-dichlorophenyldiazonium salt and the unstable components are metered exactly on the internal

according to Wahlefeld et al. [3] proven. the surface of a capillary (glass or plastic),

For this determination, a sample blank value must be taken into account. The reaction solution can be simply and easily

be viewed. Since a relatively large amount of the capillary in the plastic disposable measuring cuvette is used for the determination of bilirubin

If sample material (50 | il serum for sample blank value and analyte 15 is used. Error in dissolving and dosing the unstable se) is required, it is particularly advantageous here that sample components are excluded. The user blank value and analysis in a measuring cuvette (i.e. with 50 [d per pipetting steps are spared. This enables the analysis to be determined). perform easier, faster and more precisely.

The plastic disposable measuring cell contains 1.25 ml of the - The application of the unstable components to the internal

Except for the 2,5-dichlorodiphenyldiazonium salt, the complete surface of a capillary has particular advantages:

welded into the reagent. With a capillary the pro - Small amounts (micrograms) can be exactly in the capillary

be (e.g. 50 [xl serum) added, shaken gently and the lare introduced and handled.

Reagent blank value determined in a photometer. Connection additives are not required (e.g. fillers and the capillary, on the inner surface of which the binder is used in the manufacture of tablets), so that the 2,5-dichlorophenyldiazonium salt can be added to the cuvette. 25 additives, which are often very sensitive reactions not brought and shaken gently. After the reaction time (10 minutes.

ten) the analytical value is determined in the photometer. The lack of additives provides a quick result, the bilirubin content of the sample, is obtained by ensuring that the unstable component is dissolved (approx. 1 min),

subtracted from the analysis value the sample blank value and which during tablets e.g. 10 min until completely dissolved

Difference multiplied by a fixed factor. 30 need.

The method described under c) is preferred - turbidity, which is often caused by additives, if several become unstable in the solution for the determination and which complicate the photometric analysis,

Components are required, e.g. avoided with the enzymatic.

Cholesterol determination according to Röschlau et al. [5] with - the direct contact of dangerous substances with the skin (fin-

transmit color reaction according to Trinder [6]. 35 ger) is avoided.

In the welded plastic disposable measuring cuvette the - The shelf life of the tests is significantly extended, because there are 1.5 ml of a phosphate buffer / phenol / methanol / Hy- also unstable components of a reaction solution, dry droxypolyäthoxydodecan mixture. and stored in a cool place, often keep for years.

4-aminophenazone forms the 40 analysis time with phenol and the H202, which - The reaction time can be shortened and thus also that of the enzymatic conversion of cholesterol. For example, in hemoglobin determination

Color component (4- (p-benzoquinone monoimino) phenazone), with the hemiglobin cyanide method the pH value of the solution, which is determined photometrically. set to 7.2. This is a compromise between react

4-aminophenazone is in solid form on the inner rate of interaction, which increases with increasing pH of the solution.

surface of a glass or plastic capillary, since it decreases solution and the durability of the solution, which increases with

in solution with phenol together with the storage the pH value to be increased increases, because at a lower pH

determines the color component that, according to the enzymatic value, HCN is expelled faster from the solution and the reaction to be determined. Reaction solution quickly unusable.

The capillary coated with 4-aminophenazone is now at a pH of 7.2, the reaction time is 3

used to do this e.g. minutes suspended in ammonium sulfate.

Enzyme mixture (approx. 20 [d cholesterol esterase / cholesterol oxy so at a pH value of 6.8, the reaction time is only that / peroxidase) in the plastic disposable measuring cell for 1 minute, but the solution is no longer longer bring. The contents of the capillary time become stable by gentle shaking.

mixed with the contents of the cuvette and then the reagents. However, a blank value coated with KCN on the inside is used, determined photometrically. Now the capillary to be analyzed, with good stability you can adjust the pH of the sample (liquid, serum, plasma) with a volumetric solution to e.g. 6.8 and thus the reaction time clearly

dosed capillary (e.g. 10 [il), shorten the cuvette slightly on loan.

shakes and after a reaction time of approx. 15 minutes the - Because this cuvette analysis value is determined in the photometer for the completion of the reagents. The result that the ten-finished tests do not contain any aids (pipettes, measuring cylinders etc.) Cholesterol content of the sample can be obtained by using the rea-, these tests are ideal for subtracting the blank value from the analysis value and the differences - 60 a mobile use, such as in the rescue helicopter, emergency limit multiplied by a fixed factor. doctor's car or a home visit, especially for these tests

With the described invention it is for the first time portable, battery operated photometer (computer mini

has become possible for hematological, clinical-chemical and tometer M1000).

biochemical examinations to offer ready-to-use cuvette tests - The use of single-use plastic measuring cuvettes, which in contrast to the Mo-65 on the market, also offers price advantages. On the one hand, no additional devices, no-tests, single-glass tests or one-tests, real finished tests (pipettes, test vessels, reaction vessels) are required; Compared to the existing systems, there are always only so many cuvette-ready tests due to a whole series of sometimes surprising advantages: adding the unstable components is completed as required

This means that expensive reagents do not have to be discarded (e.g. due to insufficient stability). Thanks to the problem-free handling, errors are avoided and repeat analyzes are saved.

- Due to the problem-free handling, which largely excludes the possibility of errors, even persons who are not specially trained can carry out the analyzes and obtain correct results. As a result, these tests can also be carried out when no specially trained personnel are available (e.g. night duty, when no MTA is available, developing countries).

The possibility of instructing non-specially trained personnel to carry out these pre-cuvette tests enables further cost savings.

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Execution examples

The glass or plastic cell shown schematically in the drawing serves as the measuring cell. It is about half full with the reaction solution 1 and hermetically sealed at the top by a cap 2. The cap 2 20 also serves as a handle for the cuvette so that it is not contaminated when touched. On the cap 2 there is a pin 3 which has a predetermined breaking point 4. If you break off the pin 3 laterally, a circular hole is created in the cap surface. The capillaries 5 25 and 6 can be inserted through this hole. The capillary 5 serves e.g. for dosing the liquid sample. The capillary 6 is coated on the inside with a reaction partner in solid form. Instead of in the form of a coating, the solid reactant can fill the capillary volume even in bulk. 30 When capillaries 5 and 6 are introduced and the cuvette is shaken vigorously, the contents of capillary 5 and the reactants in capillary 6 pass into reaction solution 1. In addition, the capillaries 5 and 6 lay in the corners of the cuvette as a result of the adhesive forces and therefore do not interfere with the optical path in the photometer. Cases are conceivable in which both capillaries 5 and 6 are coated on the inside with different reactants, the capillary 5 simultaneously serving for metering. There is also the possibility that only one capillary is used 40. In this case, the capillary 5 used for dosing is also the carrier for a reaction partner.

Standard devices, such as those used e.g. are described in German Ausleschrift 2 448 206. 45

A) Hemoglobin determination using the hemoglobin cyanide method

Here, hemoglobin is oxidized to hemiglobin by potassium hexacyanoferrate (III) and this is converted into hemiglobin cyanide with potassium cyanide. 50

The measurand is the absorbance of the hemiglobin cyanide at 540 - 546 nm.

Devices: Spectrophotometer or filter photometer with measuring wavelength of 540 or 546 nm.

Reagents: Disposable plastic measuring cells with lids. 55

Layer thickness:

1.00 cm content: 1.25 ml reagent of the following composition: 0.6 mmol / 1 potassium hexacyanoferrate (III)

2.5 mmol / 1 phosphate buffer pH 7.2 (or pH 6.8) 60

1.5 mmol / 1 sodium chloride 0.05% detergent (e.g. saponin)

Volume-calibrated glass capillaries with 5 ^ 1 content (length: 32 mm; inner diameter 0.446 mm), which are coated on the inner surface with 70 [ig KCN. 65

Execution:

The predetermined breaking point 4 in the lid 2 of the plastic disposable

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The measuring cuvette is broken open using the pin 3 provided for this purpose. With the volume-calibrated glass capillary 5, e.g. from a drop of blood on the fingertip or from a sample vessel in which the blood is located, 5 (xl blood). Blood residues that may be on the outside of the capillary are stripped off and the blood-filled capillary 5 is thrown into the cuvette. The opening in the lid 2 is sealed with an adhesive label, the blood is mixed with the reagent 1 by shaking the measuring cuvette, taking care that the measuring cuvette is only touched on the lid and on the bottom, so that the cuvette walls (measuring surfaces) are not contaminated After the reaction time has elapsed (three minutes at pH 7.2, one minute at pH 6.8), the absorbance of the analysis can be measured in the photometer against a still unused plastic disposable measuring cell (= reagent blank). the hemoglobin concentration of the blood in grams of hemoglobin per 100 ml of blood is obtained by multiplying the extinction by 36.8.

Production of the KCN-coated capillaries

70 mg KCN are dissolved in 5 ml of a suitable solvent (e.g. methanol, ethanol). The capillaries 5 are filled by immersing them in this solution. The solvent is drawn off under a slight vacuum, as a result of which the KCN is distributed evenly over the inner surface of the capillary. When stored dry, these capillaries are stable for at least 12 months.

If the plastic disposable measuring cuvettes are packed impermeable to water vapor (e.g. by sealing them in deep-drawn aluminum foil), the content is also stable for at least 12 months if the volume is constant.

The measuring range and sensitivity of the determination correspond entirely to the reference method described in the DIN standard 58931.

Accuracy and precision were checked by multiple analyzes of control blood (4 C from Coulter-Counter, CH 60 from Merz and Dade). As shown in Table 1 (Appendix), the same results are obtained when determining the hemoglobin with the finished cuvettes as when determining the hemoglobin with the hemogloginometer from Coul-ter Electronics GmbH.

The regression line y = 0.9669 «X + 0.3570 (y = finished cuvette; X = hemoglobinometer) and the correlation coefficient r = + 0.9985 were calculated from the results of the comparative tests carried out on 40 human blood samples. According to these values, there is a very close correlation between the two methods.

B) Determination of bilirubin with 2,5-dichlorophenyldiazonium

salt

In this determination, the total bilirubin is coupled with 2,5-dichlorophenyldiazonium chloride to the corresponding azobiolirubin. Indirect bilirubin is released by a detergent.

The measured variable is the absorbance of the azobilirubin at 546 nm.

Devices: Spectral or filter photometer with measuring wavelength of 546 nm.

Reagents: Disposable plastic measuring cells with lids.

Layer thickness:

1.00 cm. Contents 1.25 ml reagent of the following composition: 0.1 N hydrochloric acid 1% detergent

Volume-calibrated glass capillaries with 20 | il content (length 30 mm; inner diameter 1.302 mm), which are coated on the inner surface with 2,5-dichlorophenyldiazonium chloride.

Volume calibrated glass capillaries (50 (il for dosing the sample.

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Execution:

The predetermined breaking point 4 in the lid 2 of the single-use plastic measuring cell is broken open by means of the pin 3 provided for this purpose. With a volume-calibrated glass capillary 5, 50 | il sample, e.g. Serum or plasma, introduced into the measuring cell. After the opening in the lid has been closed with the adhesive label, the sample is mixed with the reagent by gently shaking the measuring cell. It is important to ensure that the measuring surfaces are not touched. Now determine the absorbance of the measuring cell in the photometer (El5 = sample blank value). The glass capillary 6, which contains the 2,5-dichlorophenyldiazonium chloride, is then introduced into the measuring cuvette, and the cuvette is shaken gently until the 2,5-dichlorophenyidiazonium chloride has dissolved (approx. 15 seconds). After the reaction time (at least 10 minutes at room temperature), the absorbance of the analysis (E2) can be determined in the photometer.

The result, the concentration of total bilirubin in milligrams per 100 ml sample (serum or plasma), is obtained by subtracting the sample blank value (Ej) from the analysis value (E2) and multiplying the difference by 44.5.

Production of the capillaries internally coated with 2,5-dichlorophenyldiazonium chloride:

125 mg of 2,5-dichlorophenyldiazonium chloride are suspended in 10 ml of a suitable liquid (e.g. ether). Volume-calibrated glass capillaries with 20 jxl content are filled by immersing them in this solution. The liquid is drawn off under a slight vacuum. As a result, the 2,5-dichlorophenyldiazonium chloride is evenly distributed on the inner surface of the capillary. Stored dry and cool, these capillaries are stable for at least 12 months. If the plastic disposable measuring cuvettes are packed so that they are impermeable to water vapor (e.g. by sealing them in deep-drawn aluminum foil), the content is also stable for at least 12 months if the volume is constant.

The measuring range and sensitivity of the determination correspond to that of WAHLEFELD et al. (3) method described.

Accuracy and precision were checked by multiple analyzes of control sera. When determining the total bilirubin with the finished cuvettes, results are obtained which are in good agreement with the results of the determination of the total bilirubin with that of WAHLEFELD et al. (3) method described.

The regression line y = 0.9392 * X + 0.1804 (y = finished cuvette; X = test package, bilirubin DPD method from Böhringer Mannheim) and the correlation coefficient r = were calculated from the results of the comparative tests carried out on 40 samples 4- 0.9931

According to these values, there is a very close correlation between the two methods.

C) Enzymatic cholesterol determination with cholesterol esterase, cholesterol oxidase, peroxidase and subsequent color reaction after TRINDER.

In this method, cholesterol esters are cleaved by cholesterol esterase. Cholesterol is oxidized to A4 cholestenone by cholesterol oxidase. The resulting H202 is reacted with phenol and 4-aminophenazone by peroxidase to form the color component, the 4- (p-benzoquinone-monoimino) -phenazone. The measurand is the absorbance of 4- (p-benzoquinone-monoimino) -phenazone at 546 nm.

Devices: spectral or filter photometer with measuring wavelength of 546 nm.

Reagents:

Disposable plastic measuring cells with lid. Layer thickness 1.00 cm. Contents 1.50 ml of reagent with the following composition: 0.4 mmol / 1 potassium phosphate buffer pH 7.7 10 mmol / 1 phenol 1.8 mmol / 1 methanol

0.4% Hydroxypolyäthoxydodecan Volume calibrated glass capillaries with 20 | il content (length 32 mm, inner diameter 0.892), which are coated on the inner surface with 4-aminophenazone.

5 volume-calibrated glass capillaries (20 | il) for dosing the sample.

Enzyme mixture in 3.2 M ammonium sulfate solution consisting of

20 U / ml cholesterol esterase 10 6 U / ml cholesterol oxidase 4 U / ml peroxidase

execution

The predetermined breaking point in the lid of the plastic disposable measuring cell is broken open using the pin provided. With the volume-calibrated glass capillary, which is coated on the inner surface with 4-aminophenazone, 20 [xl of the enzyme mixture are introduced into the measuring cell and mixed with the contents by gentle shaking. This completes the cholesterol determination reagent. Now the extinction of the measuring cell (Ej = reagent blank) is determined in a photometer. Then, with a volume-calibrated glass capillary, 20 μl of the sample (serum or plasma) are placed in the measuring cuvette and mixed with the reagent by gently shaking it. After the reaction time (at least 10 minutes at room temperature) the absorbance of the analysis (E2) is determined in the photometer.

The result, the concentration of cholesterol in milligrams per 100 ml sample (serum or plasma) is obtained by subtracting the reagent blank (Ex) from the analysis value (E2) and multiplying the difference by 652.

Production of the capillaries coated with 4-aminophenazone:

457 mg of 4-aminophenazone are dissolved in 20 ml of a suitable liquid (e.g. dichloromethane, benzene). Volume-calibrated glass capillaries with a content of 20 fil are filled by immersing them in this solution. At approx. 60 ° C the liquid is drawn down under a slight vacuum. This distributes the 4-aminophenazone evenly on the inner surface of the 40 capillaries.

When stored cool and dry, these capillaries are stable for at least 12 months.

If the plastic disposable measuring cuvettes are impermeable to water vapor, e.g. packed by sealing in deep -45 aluminum foil, the content is stable for at least 12 months if the volume is constant.

The enzyme mixture is stored in glass vessels and is stable for at least 12 months when stored at +4 ° C.

The measuring range and sensitivity of the determination correspond to those of RÖSCHLAU et al. (5) method described.

Accuracy and precision were checked by multiple analyzes of control sera. Cholesterol determination using the finished cuvettes gives results which are in good agreement with the results of cholesterol determination using the 55 RÖSCHLAU et al. (5) agree, the test pack cholesterol CHOD-P AP method from Böehringer Mannheim was used.

The regression line y 60 = 0.9905 * X + 2.734 (y = finished cuvette; X = cholesterol test pack CHOD-PÄP method, Böehringer Mannheim) and the correlation coefficient r = + 0 were calculated from the results of the comparative tests carried out on 40 samples , 9885.

According to these values, there is a close correlation between the two methods.

65 D) Enzymatic blood sugar determination with glucose oxide, peroxidase and subsequent color reaction according to TRIN-DER (example for several sensitive components in the capillary).

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In this method, glucose is oxidized to gluconic acid by glucose oxidase. The resulting H202 is reacted with phenol and 4-aminophenazone by peroxidase to form the color component, the 4- (p-benzoquinone-monoimino) -phenazone.

The measurand is the absorbance of the 4- (p-benzoquinone-mono-imino) -phenazone at 546 nm.

Equipment:

Spectral or filter photometer with measuring wavelength of 546 nm.

Reagents:

Disposable plastic measuring cells with lid. Layer thickness 1 cm.

Contents 1.5 ml of reagent with the following composition: 0.1 mmol / 1 phosphate buffer pH 7.0 4 mmol / 1 phenol

Volume calibrated glass capillaries with 10 jxl content (length 10 mm; inner diameter 1.12 mm), which are coated on the inner surface with a mixture of glucose oxidase, peroxidase and 4-aminophenazone.

Volume calibrated glass capillaries with 10 ml content for dosing the sample.

Execution:

The predetermined breaking point in the lid of the plastic disposable measuring cell is broken open using the pin provided. The volume-calibrated glass capillary, which contains the glucose oxidase / peroxidase / 4-aminophenazone mixture, is introduced into the measuring cell, thereby completing the reagent. Immediately afterwards, 10 ml of sample (e.g. serum or plasma) are introduced into the measuring cuvette with a volume-calibrated glass capillary. The contents of the capillaries are mixed with the contents of the measuring cell by gentle shaking. The absorbance of the measuring cell (Ex = reagent or sample blank) is then determined in a photometer. After the reaction time (at least 20 minutes at room temperature) the absorbance of the analysis (E2) is determined in the photometer.

The result, the glucose concentration in milligrams per 100 ml sample (serum or plasma) is obtained by subtracting the reagent and sample blank value (Ej) from the analysis value (E2) and multiplying the difference by 597.

Production of the capillaries coated on the inside with glucose oxidase / peroxidase / 4-aminophenazone: 500 mg of glucose oxidase (degree of purity II 100 U / mg), 50 mg of peroxidase (degree of purity II 100 U / mg) and 520 mg of 4-aminophenazone in 10 ml are suitable Solvent (e.g. acetone or dichloroethane) suspended. Volume-calibrated glass capillaries of 10 | il content are filled by immersing them in this suspension. The liquid is drawn off under a slight vacuum, as a result of which the enzymes and the 4-aminophenazone are evenly distributed on the inner surface of the capillary.

When stored cool and dry, these capillaries are stable for at least 15 months.

If the plastic disposable measuring cuvettes are impermeable to water vapor, e.g. packaged by sealing in deep-drawn aluminum foil, the content of the constant volume is stable for at least 15 months.

The determination is linear in the range of 50-400 mg / glucose per 100 ml sample. The sample must be diluted at higher glucose concentrations.

Accuracy and precision were checked by multiple analyzes of control sera.

The glucose determination with the ready-to-use cuvettes gives results which are in good agreement with the results which are obtained when the automatic glucose GOD-PAP method from Böehringer Mannheim is used to determine the glucose.

From the results of the comparative examinations carried out on 40 samples, the regression grade was calculated as y = 0.9829 • X + 0.7491 (y = finished cuvette; X = vending machine package glucose GOD-PAP method, Böehringer Mannheim) and the correlation coefficient r = + 0.9923

According to these values, there is a close correlation between the two methods.

E) Enzymatic blood sugar determination using the glucose dehydrogenase method.

(Example of UV test)

In this method, β-D-glucose is converted into D-gluconolactone by the glucose-i5 dehydrogenase. NAD (nicotinamide adenine dinucleotide = coenzyme) is reduced to NADH2. The enzyme mutarotase accelerates the formation of ß-D-glucose from a-D-glucose. The measured variable is the absorbance of the NADH2 at 340 or 366 nm.

20 devices: spectral or filter photometer with measuring wavelength of 340 or 366 nm.

Reagents: Disposable plastic measuring cells with lids. Layer thickness 1 cm; Contents 1.5 ml of reagent with the following composition:

25 0.1 mmol / 1 phosphate buffer pH 7.6

Volume-calibrated glass capillaries with a volume of 10 ml (length 10 mm; inner diameter 1.12 mm), which are coated on the inner surface with a mixture of glucose dehydrogenase, mutarotase and NAD.

30 volume calibrated glass capillaries with 10 ml content for dosing the sample.

Execution:

This method determines a reagent blank (is required only once for each pack). For this purpose, a measuring cuvette with buffer is measured against a measuring cuvette with buffer and internally coated capillary. The extinction difference is the reagent blank (E0).

The sample (e.g. 10 µg serum or plasma) is placed in an open measuring cell and mixed with the buffer. Now the sample empty value is determined in the photometer (Ei). The capillary coated on the inside with glucose dehydrogenase / mu-tarotase / NAD is then introduced into the measuring cuvette and the contents are mixed with the contents 45 of the measuring cuvette by gentle shaking. After the reaction time (10 minutes at room temperature), the absorbance of the analysis (E2) is determined in the photometer.

The result, the glucose concentration in milligrams per 100 ml sample, is obtained using the following formula:

Result = E2- (Eì + E0 X 824

Production of the capillaries coated on the inside with glucose dehydrogenase / mutarotase / NAD.

100 units of glucose dehydrogenase (units), 3.8 units of mutarotase (units) and 14.6 mg of NAD

55

60 are suspended in 10 ml of a suitable solvent (e.g. dichloromethane, dichloroethane). Volume-calibrated glass capillaries with a volume of 10 ml are filled by immersion in this suspension. The solvent is drawn off under a slight vacuum, as a result of which the enzymes and the NAD 65 are evenly distributed on the inner surface of the capillary.

When stored cool and dry, the capillaries are stable for at least 15 months.

The water-impermeable packaged plastic

637 216 8

Weg measuring kivettes are also stable for at least 15 months - 150 mg of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) are in cash. The measuring range and sensitivity of the determination correspond to 10 ml of a suitable solvent (e.g. methanol or ethanol) as described by BANAUCH et al. (7) method described. suspended. Volume-calibrated glass capillaries of 10 | il Inaccuracy and precision were maintained by multiple analyzes and are filled by immersion in this suspension. Checked under by control sera. 5 slight vacuum, the solvent is removed, thereby

The glucose determination with the ready-to-use results in the acetylthiocholine iodide and the DTNB equal cuvette results, which are in good agreement with the results of the glucose on the inner surface of the capillary.

according to BANAUCH (7), whereby these capillaries are kept cool and dry.

Test pack glucose (Gluc-DH method, UV test) stable for at least 15 months.

Merck was used. io The water vapor impermeable packaged plastic

The regression could be calculated from the results of the distance measurement cuvettes carried out on 40 samples, which are also comparative tests for at least 15 months. Measuring range and sensitivity correspond to the method described by rade y = 0.9926 • X + 2.4120 FRANK (9).

(y = pre-filled cuvette; X = Merckotest ® glucose) and the correctness and precision were checked by multiple analyzes of the ratio r = + 0.9941 15 of control sera. You get at the activity

According to these values, there is a close correlation between the two methods of tuning the cholinesterase with the finished cuvettes. nisse that matched the results of cholinesterase activity

F) Determination of cholinesterase activity according to Ellman determination according to ELLMAN et al. (8) agree, whereby (example for a kinetic method) the test pack cholinesterase from Böehringer Mannheim

In the by Ellman et al. (8) described method splits 20 was applied.

the cholinesterase acetylthiocholine iodide in acetic acid and from the results of the tests carried out on 40 samples

Thiocholine iodide. Thiocholine iodide reduced the 5,5 'dithiobis equivalents, the regression line 2-nitrobenzoic acid (DTNB) calculated to 5-thio-2-nitrobenzoic acid, which y = 1.0030 • X - 0.0210

is determined photometrically at 405 nm. (y = finished cuvette; X = cholinesterase test pack from

The measured variable is the change in extinction of 5-thio-2-nitro-25 Böehringer Mannheim) and the correlation coefficient of benzoic acid at 405 nm. R = + 0.9963

Devices: Spectral or filter photometer with measuring wavelength. According to these values, there is a 405 nm between the two methods and a cuvette holder that can be heated to 25 ° C. ne close correlation

Reagents: Disposable plastic measuring cells with lids.

Layer thickness 1.00 cm; Contents 1.5 ml reagent of the following composition: Literature:

50 mmol // phosphate buffer pH 7.2 [1] C. STEFFEN:

0.1% Saponin Photometric determination of the number of erythrocytes

Volume calibrated glass capillaries with 10 jx! Contents (length Medical Lab. 5 (1959) 76 - 77 10 mm; inner diameter 1.12 mm), which on the inner upper 35

surface with a mixture of acetylthiocholine iodide and [2] L. JENDRASSIK et al. :

DTNB are coated. Biochem. Z. 297 (1938) 81

Volume calibrated glass capillaries with 5 fjl content for dosing the sample. [3] A.W. WAHLEFELD et al .:

40 scans. J. clin. Lab. Invest.

Implementation: Vol. 29, Suppl. 126 (1972) Abstract 11.12.

Since enzyme activity determinations are very temperature-dependent, the activity determination of cholinesterase [4] H. POPPER et al. :

at exactly 25 ° C. For this, the plastic disposable biochem. Z. 291 (1937) 354 measuring cell preheated to this temperature (e.g. in the temp. 45

cuvette holder of the photometer). By introducing [5] P. RÖSCHLAU et al .:

the 9th Int. coated on the inside with acetylthiocholine iodide and DTNB Congr. on clin. Chemistry,

The reagent is completed in the capillary. Then Toronto, 1975 Abstr. No. 1

with a volume-calibrated capillary 5 [d sample (e.g. serum,

Plasma or whole blood) and 50 [6] P. TRINDER:

mixed with the reagent. The measuring cuvette is in the on Ann. Clin. Biochem. 6 (1969) 24

25 ° C tempered cell holder of the photometer.

The initial absorbance (Ex) is read after 30 seconds. [7] D. BANAUCH et al. :

E2 is determined exactly 60 seconds later. Another 60 Z. Klin. Chem. Klin. Biochem.

Seconds later, E3 is determined. The extinction difference 55 13 (1976) 101 per minute is obtained as the mean of E2 - Ex and E3 - E2.

The result, the activity of cholinesterase in units per milli- [8] G.L. ELLMAN et al. :

liter (U / ml) is obtained by taking the extinction difference per biochem. Pharmacol. 7 (1961) multiplied 88 minutes (AE / min) by 22.6.

Preparation of the inside 60 [9] G. FRANK with acetylthiocholine iodide and DTNB:

coated capillaries. 2.5, g acetylthiocholine iodide and Z. analyte. Chem. 279 (1976) 155

C.

1 sheet of drawings