GB1582224A

GB1582224A - Apparatus for the preparation of a fluid for examination in optical analytic instruments

Info

Publication number: GB1582224A
Application number: GB19197/78A
Authority: GB
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 1977-05-14
Filing date: 1978-05-12
Publication date: 1981-01-07
Also published as: ATA348578A; SE7805408L; NL7805189A; FR2390724B1; AT371253B; AU3600878A; JPS6027375B2; BR7803016A; DE2721942C3; NL190427C; DE2721942A1; CH637216A5; IT7823384A0; FR2390724A1; BE866993A; AU514940B2; JPS53141688A; IT1094667B; ES469792A1; DE2721942B2

Abstract

The measuring liquid in analytical optical devices frequently has to be treated prior to the measurement. A device suitable for this purpose is based on an optical cuvette (cell) filled with a suitable reaction solution. The sample liquid is dispensed into the reaction solution with the aid of a capillary. Normally, all the reactants for the reaction are contained in the reaction solution along with the sample liquid. In the case of the device described here, however, some of the reactants are only introduced into the reaction solution using the dispensing capillary. For this purpose, the dispensing capillary, or independently thereof an additional capillary, is filled with the reactant in the solid phase. When the sample liquid and the reactant are mixed with the reaction solution in the capillary, a chemical reaction sets in which results in a characteristic change in the optical absorption. This change serves as a basis for a photometric measurement.

Description

(54) AN APPARATUS FOR THE PREPARATION OF A FLUID FOR EXAMINATION IN OPTICAL ANALYTIC INSTRUMENTS (71) We BAYER AKTENGESELLSCHAFT a body corporate organised under the laws of Germany of 509 Leverkusen, Germany do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The invention relates to an apparatus for preparing a fluid for examination in optical analytic instruments and is based on a sealed vessel filled with an accurately prepared reaction solution and a capillary tube which may introduced into the vessel for dispensing the sample fluid into the reaction solution. The invention is applicable in particular to the analytical examination of body fluids.

"Ready tests" are increasingly offered in the sphere of haematological, clinico-chemical and biochemical investigations and these "ready tests" allow the user to rapidly and easily determine haematological, clinico-chemical and bio-chemical parameters without even having to add reagents.

These "ready tests", also known as "monotests", "single-tests" or "single glass tests", are characterised in that unstable components of the reagent which is ready for use may be stored usually in freeze-dried form in glass or plastic containers.

Before the reagent is ready for use, a solvent which may contain still further substances required for the complete reagent is added.

In some cases, for example in the determination of haemoglobin, the reagent, which is ready for use, may be present in the glass or plastic container.

The sample to be analysed e.g. fluid, blood, plasma or serum, is then added to the prepared reagent usually by means of a pipette so as to guarantee accurate dispensing. The reagentsample mixture is subsequently conveyed to a measuring vessel, usually a glass or quartz, and is analysed in a measuring instrument, for example a photometer.

Sealable vessels have recently been disclosed (German Offenlegungsschrift No. 2,422,260) which are designed as plastics single use measuring vessels (preferably composed of polystyrene) and are hermetically sealed by means of a plastic cover.

In this case, the cover is joined to the vessel by bonding, for example, by means of ultrasonics during mass production. The cover has a predetermined point for fracture, which may be broken by means of a pin. The sample to be analysed may be introduced into the vessel through the opening thus produced in the vessel cover.

The vessel itself already contains the complete reagent fluid. In photometric determination fo the erythrocytes number, for example, it is only necessary for the user to introduce 5 ul of blood into the vessel through the opening in the cover using a volume-calibrated glass capillary tube.

Photometric measurement may take place as soon as the blood has mixed completely with the reagent. This method is particularly good because the user has neither to add nor to dissolve the reagent, nor does he require auxiliary instruments for the analysis such as pipettes. This simplifies analysis considerably so that it may be carried out correctly by people who have not been specially trained.

The previously known methods described have the following disadvantages: During the "ready tests" it is still usually necessary to make transfers using a pipette.

Expensive and sensitive auxiliary instruments such as, for example, Eppendorf-Pipettes are required for this purpose. The using of a pipette itself represents a source of error.

The solvent required, for example, distilled water, is not always available in the quality demanded (for example quartz-distilled water).

The complete reagent or the complete reagent-sample mixture still has to be conveyed into a measuring vessel, for example a glass vessel, before the actual measuring can take place. This is an additional operating stage which provides the further possibility the introduction of errors, such as, for example, vessels which are not completely clean or not completely dry (producing errors in volume) or residues or detergents in the vessels. In all, the carry8ing out of these "ready tests" is so complicated that they may not justifiably be called "ready tests" and there is such a large number of sources of error that these cannot be carried out by people who have not been specially trained.

The test described in German Offenlegungsschrift No. 2,422,260 substantially meets the requirements for a "ready test". The user neither has to dissolve reagents nor has he to mix various solutions exactly or to convey the prepared reagent-sample mixture to a measuring vessel. He merely has to introduce into the vessel the accurately measured sample to be analysed and this may be carried out easily, for example by means of volumecalibrated glass capillary tubes, and the vessel may then be conveyed to the photometer.

Possible sources of error which may arise in such operations are thus eliminated from the beginning and the process of analysis is simplified in such a way that even untrained personnel are able to carry out analysis correctly.

Unfortunately, there are, however, only a few reagents for biochemical, haematological and clinico-chemical analyses which remain for prolonged periods (6 months) in a usable state.

This applies in particular to the modern enzymatic methods, as for example, for determining the blood sugar level using glucoseoxidase/peroxidase, hexokinase/glucose-6phosphate-dehydrogenase or glucose-dehydrogenase-mutarotase, for the enzymatic determination of uric asid, urea, triglyceride and cholesterol and, of course, for determining the activity of enzymes themselves.

However, reagents for non-enzymatic determination often do not remain usable over a prolonged period (6 months). This applies, for example, in the determination of bilirubin using diazotized sulphanilic acid according to Jendrassik and Grof [2] or using 2,5-dichlorophenyl-diazonium salt according to Wahlefeld et al [3], similarly in determining creatinin using picric acid/NaOH according to Popper petal [4] .

The use of single use measuring vessels is accompanied by further problems. Glass is ruled out as material for the single use measuring vessels since glass vessels are much too expensive in the quality required for photometric measurements.

The use of plastics, for example polystyrene, does allow inexpensive mass production of the single use measuring vessels but is frequently accompanied by problems with regard to the durability of the reagents.

Polystyrene is not absolutely impermeable to water vapour and if no suitable premature means are found, the volume of the reagent fluid may alter in the sealed vessel during the storage period. In view of the fact that the analyses to be carried out are dependant upon the mixing ratio being accurately known, false results would thus be obtained.

Polystyrene is also permeable to many gases.

This would be detrimental, for example to the durability of the reagent for determining haemoglobin as haemiglobin cyanide. This reagent contains, in addition to a buffer substance and a detergent for haemolysis of the erythrocytes, pottassium hexacyanoferrate (III), which oxidises haemoglobin to haemiglobin and KCN which converts the haemiglobin into haemiglobin cyanide. The haemiglobin cyanide is subsequently determinec photometrically.

The KCN contained in the reagent is in equilibrium with HCN in the volume of gas above the fluid. HCN may however diffuse through the polystyrene wall of the vessel so that the CN content of the reagent decreases with a half-life period of about 4 weeks. The reagent is not therefore sufficiently durable for marketing.

An object of the invention is accordingly, to overcome the problems of service life described above and to develop "ready tests" with which ready-prepared plastic single use vessels may be used allowing the user to obtain correct analytical results in a short period without additional instruments, as for example, pipettes and without special training.

According to the invention there is provided an apparatus for preparing a fluid for examination in optical analytic instruments comprising a sealed vessel containing an accurately prepared reaction solution and a capillary tube which is insertable into the vessel for dispensing a sample fluid into the reaction solution, wherein in order to regulate the optical properties of the fluid for examination at least one of the reactants required for the formation of the said fluid for examination from the said reaction solution is located in the solid phase in the said capillary tube used for dispensing the sample fluid and/or in a second capillary tube for introduction into the vessel.

The reactants are preferably placed on the internal wall of the capillary tube in a finely divided form.

In this way, the spectrum of the reactants may be enlarged considerably. Frequently, all the reactants cannot be stored together in one solution since they become unstable within a short period or react with each other in this form. This problem is neatly solved by storing such unstable components in the capillary tubes. Reactions previously considered unsuitable for the analytical process described in German Offenlegungsschrift No. 2,422,260 owing to the unstability of the reactants may thus be achieved. The capillary tubes coated with one reactant and stored dry may remain stable for long periods (more than 6 months) while the reactants which are stable in the solution may be accurately prepared and fed into the measuring vessel.

The technician can now: a) Take up the sample )fluid, blood, plasma, serum) with the capillary tube coated on its internal surface and introduce the filled capillary tube into the plastics single use vessel. The complete reagent has thus been formed in a very simple and rapid operating stage and the sample to be analysed has been dispensed accurately. After a reaction period, if required, the sample may be analysed in a measuring instrument (for example a photometer).

In this case, the capillary tube must, of course, be calibrated in volume and the component of the reagent applied to the internal surface of the capillary tube must not falsify the full volume.

b) Introduce the capillary tube coated on the internal surface into the ready prepared plastics single use measuring vessel thus forming the complete reagent. The sample to be analysed (fluid, blood, plasma, serum) may then be added (for example using a volume-calibrated capillary tube). After a reaction period, if required, the sample may be analysed in a measuring instrument (for example, a photometer). If a control value for the reagent has to be determined during the test, then this may be obtained before adding the sample to be analysed.

If a control value for the sample is to be determined, it is preferable if the sample is introduced first into the ready prepared plastics single use measuring vessel, the control value is determined, the complete reagent is formed by adding the coated capillary tube and then the analytical value is determined. Both the sample control value and the analytical value may thus be determined in one plastics single use measu ring vessel. This saves the use of an extra vessel and that amount of sample which would otherwise be necessary for determin ing the sample control value.

c) Take up another unstable component, for example an enzyme suspension or the suspension of an enzyme mixture, using the capillary tube coated on its internal surface, and introduced this into the ready prepared plastics single use measuring vessel thus forming the complete reagent.

The sample may be added before or after adding the unstable components depending upon whether a reagent control value or a sample control value has to be determined.

The process described under (a) has proved particularly successful, for example, when determining the haemoglobin as haemoglobin cyanide.

1.25 ml of the reagent which is complete apart from the KCN are fed into the plastics single use measuring vessel. The KCN (70 ,ug per capillary tube) is located, in a finely divided state, on the internal surface of a 5 ul capacity volume-calibrated capillary tube. The sample, blood in this case, is taken up by this capillary tube. The capillary is automatically filled by capillary action as soon as it is placed in contact with the blood. The KCN dissolves immediately in the blood and does not measurably affect the accuracy of sample dispensing. Once the capillary tube has filled with blood, it is introduced into the plastics single use measuring vessel. The contents of the capillary tube are mixed with the contents of the vessel by lightly shaking the measuring vessel and the reagent is simultaneously completed.

After a reaction period of 3 minutes, the haemoglobin content of the sample may be determined directly in a photometer.

The method described under (b) has proved particularly successful when determining bilirubin using 2,5-dichlorophenyl-diazonium salts according to Wahleseld et al [3] . During this determination, a sample control value must be taken into consideration. Since a relatively large quantity of sample material is required for determining the bilirubin 50 ijl of serum each content (the sample control and the analysis volumes requiring it is particularly adventageous for the sample control value and analysis value determinations to be adapted so that they can be obtained in one measuring vessel (i.e. with 50 ul of sample).

1.25 ml of reagent which is complete except for the 2,5-dichlorodiphenyl diazonium salt are fed into the plastics single use measuring vessel. The sample (for example 50 ul of serum) is added to the vessel using a capillary tube, and the vessel is lightly shaken. The reagent control value can then be determined in a photometer. The capillary tube on whose internal surface is located the 2,5-dichloropheny diazonium salt, is subsequently introduced into the vessel and it is lightly shaken. The analytic value is determined in the photometer after the reaction period (10 minutes). The result, the bilibubin content of the sample, is obtained by subtracting the sample control value from the analytical value and multiplying the difference by a fixed factor.

The method described under (c) is preferably used if several components required for the determination are unstable in solution, as for example, in the enzymatic cholesterol determination according to Rschlau et al. [5] with subsequent colour reaction according to Trinder [6]. 1.5 ml of a phosphate buffer/phenol/methanol/hydroxy-polyethoxy dodecane mixture are located in the sealed plastics single use measuring vessel.

4-amino phenazone reacts with phenol and with the H2 02, formed during the enzymatic reaction of the cholesterol to give a colour component (4-(p-benzoquinone-monoimino) phenazone), which is determined photometrically.

4-amino phanazone is located in solid form on the internal surface of a glass or plastics capillary tube, since it forms in solution with phenol, even during storage,the colour component to be determined after the enzymatic reaction.

The capillary tube coated with 4-amino phenazone is now used for introducing the enzyme mixture suspended in, for example, ammonium sulphate (about 20 ul of cholesterol esterase/cholesterol oxidase/peroxidase) into the plastics single using measuring vessel. The contents of the capillary tube are mixed with the contents of the vessel by light shaking and the regent control value is then determined photometrically. The sample to be analysed (fluid, serum, plasma) is now added with a volume-calibrated capillary tube, (for example 10 ,us), the vessel is lightly shaken and the analytical value determined in the photometer after a reaction period of about 15 minutes.

The result, the cholesterol content of the sample, is obtained by subtracting the reagent control value from the analytical value and by multiplying the difference by a fixed factor.

The invention described has made it possible for the first time to offer vessel "ready tests" for haemotological, clinico-chemical and biochemical investigations which, unlike the commercially available "mono-tests", "single glass tests" or "single tests" are actual "ready tests".

The use of measuring vessels of the invention allows the same vessel to be used for preparing the sample and for measurement. The user therefore no longer needs to mix the sample with the reagent in a special reaction vessel and to subsequently convey it to the measuring vessel.

This means both a saving in cost (no reaction vessels required) and a simplification in operation.

Since the measuring vessels contain all liquid constituents of the reaction solution, in exact quantities and the unstable components are located, in exact quantities, on the internal surface of a capillary tube (glass or plastic), the reaction solution may be completed easily and simply by inserting the capillary tube into the plastics single use measuring vessel. Errors when measuring out and dissolving the unstable components are thus eliminated. The user does not have to carry out stages using a pipette.

Analysis may thus be carried out more simply, more rapidly and more accurately.

The applying of the unstable components to the internal surface of a capillary tube has particular advantages: Small amounts (micrograms) may be introduced exactly into the capillary tube and contained.

Additives are not required (for example fillers and binders required in the production of tablets) which might interfere with the reactions which are frequently sensitive to additives.

The unstable component is guaranteed to dissolve rapidly (about 1 minute) since there are no additives, while tablets require, for example, 10 minutes for complete dissolution.

Turbidity is often produced by additives and renders photometric analysis more difficult is avoided.

Dangerous materials are prevented from coming into direct contact with the skin (fingers).

The durability of the tests is considerably increased since even unstable components in a reaction solution, when stored dry and cool, often last for years.

The reaction period may be reduced and thus also the analysis period. Thus, for example, when determining the haemoglobin by the haemoglobin cyanide method, the pH value of the solution is regulated to 7.2. This is a compromise between the reaction velocity which decreases as the pH value of the solution increases and the durability of the solution which increases as the pH value increases because HCN is driven out of the solution more rapidly at a lower pH value and the reaction solution therefore rapidly becomes unusable.

The reaction period amounts to 3 minutes at a pH value of 7.2.

At a pH value of 6.8 the reaction period only amounts to 1 minute but the solution is no longer stable for a prolonged period.

However, if a capillary tube coated internally with KCN is used, then the pH value of the solution may be regulated to, for example, 6.8 with good stability and the reaction period may thus be clearly reduced.

Since no auxiliary agents such as pipettes or measuring cylinders are required for completing the reagents of these vessel ready tests, these tests are ideally suited to a mobile use, for example, in a rescue helicopter, ambulance or during home visits, particularly since a portable battery-operated photometer (Compur mini-photometer M 1000) is available for these tests.

The use of the plastics single use measuring vessels also affords an advantage with regard to cost. On the one hand, no additional instruments such as pipettes, sample containers or reaction containers are required and, on the other hand, only quantity of that measuring fluid required for the vessel "ready test'' is formed by adding the unstable component and thus no expensive reagents have to be rejected (for example, owing to lack of stability).

As a result of the simplified operation errors are avoided and it is therefore not necessary to carry out repeat analyses.

Owing to the simplified operation which substantially reduces the possibilities of error, people who have not been specially trained may also carry out and obtain correct results for the analyses. These tests may thus also be carried out if there are no specially trained personnel available (for example, during night duty when there are no medical technical employees available, in developing countries).

The opportunity to have people who have not been specially trained to perform these vessel "ready tests" allows further financial saving.

With reference to the accompanying drawings.

A vessel composed of glass or plastic shown diagrammatically in the drawing serves as a measuring vessel. It is filled about half full with a reaction solution 1 and is hermetically sealed at its upper end by a cap 2. The cap 2 serves at the same time as a handle for the vessel so that it is not contaminated when being grasped. There is a pin 3 with a set point of fracture 4 on the cap 2. If the pin 3 is broken off laterally, then a circular hole is produced in the surface of the cap. Capillary tubes 5 and 6 may be inserted through this hole. The capillary tube 5 serves, for example, for dispensing the fluid sample. The capillary tube 6 is coated on its inner face with a reactant in solid form. Instead of taking the form of a coating, the solid reactant may also fill out the volume of the capillary tube in a loose fill.

When the capillary tubes 5 and 6 are introduced and the vessel is shaken vigorously, the contents of the capillary tube 5 and the reactant in the capillary tube 6 are transferred to the reaction solution 1. The capillary tubes 5 and 6 are also brought into the corners of the vessel as a result of the forces of adhesion and do not therefore interfere with the optical path of rays in- the photometer. It is conceivable for both capillary tubes 5 and 6 to be coated internally with various reactants, in which case the capillary 5 is at the same time used for dispensing the sample. There is, furthermore, the possibility of operating with only one capillary tube. In this case, the capillary tube 5 used for dispensing the sample is at the same time the carrier for a reactant.

Commercial instruments of the type described, for example, in German Auslegeschrift No. 2,338,206 may be used as the photometer.

EXAMPLES A)Determination of haemoglobin by the haemoglobin cyanide method In this case, haemoglobin is oxidised to haemiglobin by means of potassium hexacyan oferrate (III) and the haemiglobin is con verted into haemiglobin cyanide by means of potassium cyanide.

The quantity to be measured is the extinction produced by the haemiglobin cyanide at a wavelength from 540 to 546 nm.

Instruments: Spectral or filter photometer with measuring wavelength of 540 or 546 nm.

Reagents: Plastics single use measuring vessels with lid.

Thickness of layer: 1.00 cm; content: 1.25 of reagent having the following composition: 0.6 mmol/l of potassium hexacyanoferrate (III) 2.5 mmol/l of phosphate buffer pH 7.2 (or pH 6.8) 1.5 mmol/l of sodium chloride 0.05% of detergent (for example, Saponin) Volume-calibrated glass capillary tubes with 5 p1 content (length: 32 mm; internal diameter 0.446 mm), which are coated in ternally with 70 pg of KCN.

Method The set point of fracture 4 in the lid 2 of the plastics simple use measuring vessel is broken by means of the pin 3 provided for that purpose. 5 pl of blood are taken, for example, from a drop of blood from the fingertip or from a sample vessel containing the blood by means of the volume-calibrated glass capillary tube. Any blood which may remain on the exterior of the capillary tube is stripped off and the blood-filled capillary tube 5 is inserted into the vessel. The opening formed in the lid 2 is sealed by an adhesive label. The blood is mixed with the reagent 1 by shaking the measuring vessel, taking care that the measuring vessel is handled only on the lid and on the base so that the walls of the vessel (measuring faces) are not contaminated. After the reaction period (three minutes at pH 7.2, one minute at pH 6.8) the extinction produced by the analysis may be compared with that obtained from a plastics single use measuring vessel which has not yet been used (= reagent control value) in the photometer. The result, the haemoglobin concentration of the blood, is obtained in grams of haemoglobin per 100 ml of blood by multiplying the extinction by 36.8 Production of the capillary tubes coated internally with KCN 70 mg of KCN are dissolved in 5 ml of a suitable solvent (for example methanol or ethanol). The capillary tubes 5 are filled by immersion in this solution. The solvent is drawn off under a slight vacuum and the KCN is thus evenly distributed on the internal surface of the capillary tube. When stored dry, the contents of these capillary tubes are stable for at least 12 months.

If the plastics single use measuring vessels are packed so as to be impermeable to water vapour (for example, by sealing in deep-drawn aluminium foil), then their contents are also stable for at least twelve months with constant volume.

The range of measurement and sensitivity of determination correspond completely to the reference method described in the German Provision DIN Standard No. 58931.

The correctness and accuracy of measuremen were checked by analysing control blood several times (4 C of Coulter-Counter, CH 60 of Merz and Dade). As shown in Table 1 (appendix), the same results were obtained when determining the haemoglobin using the "ready" vessels of this invention as when determining the haemoglobin using the haemoglobinometer produced by Coulter Electronics GmbH.

The regression line y=0.9669 x + 0.3570 (y = "ready" vessel;x = haemoglobinometer) and the coefficient of correlation r = + 0.9985 were calculated from the results of the comparative experiment carried out on 40 samples of human blood. According to these values, there was a very close correlation between the two methods.

B) Determination of bilirubin using 2,5-dichloro phenyl-diazonium salts.

In this determination, the total bilirubin is coupled with 2,5-dichloropheynl diazonium chloride to form the corresponding azobilirubin.

Indirect bilirubin is liberated by a detergent.

The quantity to be measured is the extinction, produced by azobilirubin at a wavelength of 546 nm.

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

Reagents: Plastics single use measuring vessels with lid.

Thickness of layer: 1.00 cm; Content: 1.25 ml of reagent having the following com position: 0.1 N of hydrochloric acid 1% of detergent Volume-calibrated glass capillary tubes with 20 pl content (length 30 mm; internal diameter 1.302 mm), coated on the internal surface with 2,6-dichlorophenyl diazonium chloride.

Volume-calibrated glass capillary tubes (50 /11) for dispensing the sample.

Method The desired product of fracture 4 in the lid 2 of the plastics single use measuring vessel is broken by means of the pin 3 provided for this purpose. 50 /ll of sample, for example, serum or plasma, are introduced into the measuring vessel using a volume-calibrated glass capillary tube 5. Once the opening in the lid has been sealed with the adhesive label, the sample is mixed with the reagent by lightly shaking the measuring vessel. In so doing, care must be taken that the measuring faces are not touched. The extinction obtained for the measuring vessel (E1 = sample control value) is now determined in the photometer.

The glass capillary tube 6 containing the 2,5-dichlorophenyl diazonium chloride is subsequently introduced into the measuring vessel and the vessel is shaken lightly until the 2,5-dichlorophenyl diazonium chloride has dissolved (about 15 seconds). After the reaction period (at least 10 minutes at room temperature) the extinction for the analysis (E2) is determined in the photometer.

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

Production of the capillary tubes coated internally with 2,5-dichlorophenyldiazon ium chloride 125 mg of 2,5-dichlorophenyl diazonium chloride are suspended in 10 ml of a suitable fluid (for example ether). 20 pl volumecalibrated glass capillary tubes are filled by immersion in this solution. The fluid is drawn off under a slight vacuum. The 2,5-dichlorophenyl diazonium chloride is th of reagent having the following composition: 0.4 mmol/l of potassium phosphate buffer pH 7.7 10 mmol/l of phenol 1.8 mmol/l of methanol 0.4% of hydroxypolyethoxydodecane 20 pal volume-calibrated glass capillary tubes (length 32 mm, internal diameter 0.892), coated on the internal surface with 4-amino phenazone. Volume-calibrated glass capillary tube (20 pI) for dispensing the sample.

Enzyme mixture in 3.2 M of ammonium sulphate solution composed of 20 U/ml of cholesterol esterase 6 U/ml of cholesterol oxidase 4 U/ml of peroxidase Method The set point of fracture in the lid of the plastics single use measuring vessel is broken by means of the pin provided for this purpose.

20 pal of the enzyme mixture are introduced into the measuring vessel using the volumecalibrated glass capillary tube coated with 4aminophenazone on the internal surface, and are mixed with the contents by light shaking.

The complete reagent for determination of the cholesterol is thus formed. The extinction produced by the measuring vessel (El = reagent control value) is now determined in a photometer. 20 pl of the sample (serum or plasma) are subsequently introduced into the measuring vessel using a volume-calibrated glass capillary tube and mixed with the reagent by light shaking. Upon completion of the reaction period (at least ten minutes at room temperature), the extinction for the analysis (E2) is determined in the photometer.

The result, the concentration of the cholesterol, is determined in milligrams per 100 ml of sample (serum or plasma) by subtracting the reagent control value (E1) from the analysis value (E2) and multiplying the difference by 652.

Production of the capillary tube coated internally with 4aminophenazone 457 of 4-aminophenazone are dissolved in 20 ml of a suitable fluid (for example dichloromethane or benzene). 20,us volume-calibrated glass capillary tubes are filled by immersion in this solution. The fluid is drawn off under a slight vacuum at 600 C. The 4-aminophenazone is thus evenly distributed on the internal surface of the capillary tubes.

The contents of these capillary tubes are stable for at least 12 months if stored cool and dry.

If the plastics single are measuring vessels are impermeable to water vapour, for example by sealing in deep-drawn aluminium foil, then the contents are stable for at least 12 months with constant volume.

The enzyme mixture is stored in glass containers and is stable for at least 12 months if stored at +40 C.

The range of measurement and sensitivity of determination corresponds to the method described by Röschlau et al. (5).

The correctness and accuracy of measurement were checked by analysing control sera several times. When determining the cholesterol using the ready vessels, results which compared well with the results of cholesterol determination by the method according to Röschlau et al. (5) were obtained, wherein the test packaging cholesterol CHOD PAP-method according to Boehringer Mannheim was used.

The regression lines y = 0.9905 x + 2.734 (r = ready vessel; x = cholesterol CHOD-PAP method test package, Boehringer Mannheim) were calculated from the results of the comparative experiments carried out on 40 samples and the coefficient of correlation was r = + 0.9885.

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

D) Enzymatic determination of blood sugar using glucose oxidase, peroxidase and subsequent colour reaction according to Trinder (example for several sensitive components in the capillary tube).

With this method, glucose is oxidised by glucose oxidase to form gluconic acid. The H202 thus formed is reacted with phenol and 4-aminophenazone using peroxidase to form the colour component, 4-(p-benzoquinone-monoimino)-phenazone.

The quantity to be measured is the extinction produced by the 4-(p-benzoquinone-monoamino)-phenazone at a wavelength of 546 nm.

Instruments: Spectral of filter photometer with measuring wavelength of 546 nm.

Reagents: Plastics single use measuring vessels with lid Thickness of layer: Icm.

Contents: 1.5 ml of reagent having the following composition: 0.1 mmol/l of phosphate buffer pH 7.0 4 mmol/l of phenol 10 pl volume-calibrated glass capillary tubes (length 10 mm; internal diameter 1.12 mm), coated on the internal surface with a mixture composed of glucose oxi dase, peroxidase and 4-aminophenazone.

10 It volume-calibrated glass capillary tubes for dispensing the sample.

Method The set point of fracture in the lid of the plastics single use measuring vessel is broken by means of the pin provided for this purpose.

The volume-calibrated glass capillary tube containing the mixture of glucose oxidase, peroxidase and 4-aminophenazone is introduced into the measuring vessel and the complete reagent is thus formed. Immediately afterwards, 10 p1 of sample (for example serum or plasma) are introduced into the measuring vessel using a volume-calibrated glass capillary tube. The contents of the capillary tubes are mixed with the contents of the measuring vessel by light shaking. The extinction produced by the measuring vessel (E1 = reagent control value and sample control value) is subsequently determined in a photometer. Upon completion of the reaction period (at least 20 minutes at room temperature), the extinction for the analysis (E2)-is determined in the photometer.

The result, the glucose concentration, is obtained in milligrammes per 100 ml of sample (serum or plasma) by subtracting the reagent control valve and sample control value (E1) from the analytical value (E2) and by multiplying the difference by 597.

Production of the capillary tubes coated intemally with glucose oxidase, peroxidase, 4-aminophenazone 500 mg of glucose oxidase (degree of purity If 100 U/mg), 50 mg of peroxidase (degree of purity If 100 U/mg) and 520 mg of 4-aminophenazone are suspended in 10 ml of a suitable solvent (for example acetone or dichloroethane).

10,us volume-calibrated glass capillary tubes are filled by immersion in this suspension. The fluid is drawn off under a slight vacuum and the enzyme and the 4-aminophenazone are thus evenly distributed on the internal surface of the capillary tube.

The contents of these capillary tubes are stable for at least 15 months if stored cool and dry.

If the plastics single use measuring vessels are packed so as to be impermeable to watervapour, for example by sealing in deep-drawn aluminium foil, then the contents are stable for at least 15 months with constant volume.

The determination is linear in the range of from 50 to 400 mg of glucose per 100 ml of sample. The sample has to be diluted at higher glucose concentrations.

The correctness and accuracy of measurement are checked by analysing control sera several times.

When determining glucose using the ready vessels, results were obtained which compare well with the results obtained when using the automated packing glucose GOD-PAP method of Boehringer Mannheim for determining the glucose.

The regression line y = 0.9828 x + 0.7491 (y = ready vessel; x = automatic glucose GOD PAP package method, Boehringer Mannheim) and the coefficient of correlation r = + 0.9923 are calculated from the result of the comparative experiments carried out on 40 samples.

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

E) Enzymatic determination of blood sugar by the glucose - dehydrogenase method (example of W test) With this method, ss-D-glucose. is converted into D-gluconolactone by the glucose-dehydrogenase. In so doing, NAD (nicotinamide adenine dinucleotide = co-enzyme) is reduced to NAPS2. The enzyme mutarotase accelerates the formation of the ss-D-glucose from a-Dglucose. The quantity to be measured is the extinction for the NADH2 at a wavelength of 340 or 366 nm.

Instruments: Spectral or filter photometer with measuring wavelength of 340 or 366 nm.

Reagents: Plastics single use measuring vessels with lid.

Thickness of layer: 1 cm; content 1.5 ml of reagent having the following composition: 0.1 mmol/l of phosphate buffer pH 7.6 10 pal volume-calibrated glass capillary tubes (length 10 mm; internal diameter 1.12 mm), coated on the internal surface with a mixture composed of glucose-dehydrogenase, mutarotase and NAD.

10 pal volume-calibrated glass capillary tubes for dispensing the sample.

Method With this method, a reagent control value (required only once for each packing) is determined. For this purpose, a measuring vessel with buffer is measured against a measuring vessel with buffer and internally coated capillary tube. The extinction difference is the reagent control value (ego).

The sample (for example 10,us of serum or plasma) is introduced into an open measuring vessel and is mixed with the buffer. The sample control value is now determined (E, ) in the photometer. The capillary tube coated internally with glucose dehydrogenase, mutarotase, NAD is subsequently introduced into the measuring vessel and the content is mixed with the content of the measuring vessel by light shaking. Upon completion of the reaction period (10 minutes at room temperature), the extinction produced by the analysis (E2) is determined in the photometer.

The result, the glucose concentration in milligrammes per 100 ml of sample, is obtained by the following formula: Result=E2-(E1 + E,)x 824 Production of the capillary tubes coated internally with glucose dehydrogenase, mutarotase, NAD 100 units of glucose dehydrogenase 3.8 units of mutarotase and 14.6 mg of NAD are suspended in 10 ml of a suitable solvent (for example dichloromethane or dichloroethane). 10 pal volume-calibrated glass capillary tubes are filled by immersion in this suspension.

The solvent is drawn off under a slight vacuum and the enzymes and the NAD are evenly distributed on the internal surface of the capillary tube.

The contents of these capillary tubes are stable for at least 1 5 months if stored cool and dry.

The contents of the plastics single use measuring vessels, stored so as to be impermeable to water-vapour, also remain usable for at least 15 months. The measuring range and the degree of sensitivity of the determination are comparable with the method described by Banauch et awl (7).

The correctness and accuracy of measurement were checked by analysing control sera several times.

In determining the glucose content using the ready vessels, results were obtained which compared well with the results obtained in glucose determination according to Banauch (7), wherein the test packing glucose (Gluc DH method, LV test of Merck was used.

The regression line y = 0.9926 x + 2.4120 (y = ready vessel; x = Merckotest (registered Trade Mark) Glucose) and the coefficient of correlation r = + 0.9941 were calculated from the results of the comparative experiments carried out on 40 samples.

F) Determination of the choline esterase activity by the method according to Ellman (Example for a kinetic method) With the method described by Ellman et al (8), the choline esterase splits acetyl thiocholine iodine into acetic acid and thiocholine iodide.

Thiocholine iodide reduces the 5,5'-dithiobis2-nitrobenzoic acid (DTNB) to 5-thio-2-nitrobenzoic acid which is determined photometrically at a wavelength of 405 nm.

The quantity to be measured is the change in extinction produced by the 5-thio-2-nitrobenzoic acid at a wavelength of 405 nm.

Instruments: Spectral or filter photometer with measuring wavelength of 405 nm and vessel holder temperature-adjustable to 25 CC.

Reagents: Plastics single use measuring vessel with lid. thickness of layer: 1.00 cm; contents: 1.5 ml of reagent having the following composition: 50 mmol/l of phosphare buffer pH 7.2 0.1% of Saponin 10 pal volume-calibrated glass capillary tubes (length 10 mm; internal diameter 1.12 mm), which are coated on their internal surface with a mixture of acetyl thiocholine iodide and DTNB.

5 pl volume-calibrated glass capillary tubes for dispensing the sample.

Method Since enzyme activity determination are very markedly dependent upon temperature, the activity of the choline esterase must be determined at exactly 250C. For this purpose, the plastics single use measuring vessel is preheated to this temperature (for example in the temperature-adjustable vessel holder of the photometer) The complete reagent is formed by introducing the capillary tube coated internally with acetyl thiocholine iodide and DTNB. 5 pal of sample (for example, serum, plasma, or whole blood) are subsequently added using a volume-calibrated capillary tube and are mixed with the reagent by light shaking.

The measuring vessel is placed in the vessel container of the photometer, set at a temperature of 25"C. The initial extinction (E1) is read off after 30 seconds.

E2 is determined exactly 60 seconds later.

E3 is determined another 60 seconds later.

The extinction difference per minute is obtained as average value of E2-E1 and E3-E2 - The result, the activity of the choline esterase in units per millilitre (U/ml), is obtained by multiplying the extinction difference per minute (E E/min) by 22.6.

Production of the capillary tubes coated internally with acetyl thiocholine iodide and DTNB 2.25 g of acetyl thiocholine iodide and 150 mg of 5,5'-dithiobis-2-nifrobenzoic acid (DTNB) are suspended in 10 ml of a suitable solvent (for example methanol or ethanol).

10 pal volume-calibrated glass capillary tubes are filled by immersion in this suspension. The solvent is drawn off under a slight vacuum and the acetyl thiocholine iodide and the DTNB are thus evenly distributed on the internal surface of the capillary tube.

The plastics single use measuring vessel packed so as to be impermeable to watervapour also lasts for at least 15 months.

The measuring range and sensitivity correspond to the method described by Frank (9).

The correctness and precision were checked by analysing control sera several times. When determining the activity of the choline esterase using the ready vessels, results were obtained which compared well with the results of the choline esterase activity by Ellman et al (8), wherein the choline esterase test package of Boehringer Mannheim was used.

The regression line y = 1.0030, - 0.0210 (y = ready vessels; x = test packaging choline esterase of Boehringer Mannheim) and the coefficient of correlation r = + 0.9963 were calculated from the results of the comparative experiment carried out on 40 samples.

LITERA TURE: [1] C. STEFFEN: Photometrische Bestimmung der Eryth orzytenzahl Arztl. Lab. 5 (1959) 76-77 [2] L. JENDRASSIK et al.: Biochem. Z. 297(1938)81 [3] A.W. WAHLEFELD et al.: Scand. J. clin. Lab. Invest.

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

[4] H. POPPER et al.: Biochem. Z. 291 (1937) 354 [5] P. RÖSCHLAU et al.: 9th Int. Congr. on Clin. Chemistry, Toronto, 1975 Abstr. No. 1 [6] P. TRINDER: Ann. Clin. Biochem. 6 (1969) 24 [7] D. BANAAUCH et al.: -Z. Klin, Chem. Klin, Biochem.

13(1976)101 [8] Gt. ELLMAN et al.: Biochem. Pharmacol. 7(1961) 88 [9] G. FRANK: Z. Analyt. Chem. 279 (1976) 155 WHAT WE CLAIM IS: 1. An apparatus for preparing a fluid for examination in optical analytic instruments comprising a sealed vessel containing an accurately prepared reaction solution and a capillary tube which is insertable into the vessel for dispensing a sample fluid into the reaction solution, wherein in order to regulate the optical properties of the fluid for examination at least one of the reactants required for the formation of the said fluid for examination from the said reaction solution is located in the solid phase in the said capillary tube used for dispensing the sample fluid and/or in a second capillary tube for introduction into the vessel.

2. An apparatus according to Claim 1, wherein the said at least one reactant is applied to the internal wall of the capillary tube, in a finely divided form.

3. An apparatus according to Claim 1 or 2, wherein for determining the haemoglobin concentration of a sample the reaction solution in the vessel comprises a phosphatebuffered potassium hexacyanoferrate (III)/ sodium chloride solution and the capillary tube used for dispensing the sample is coated with potassium cyanide.

4. An apparatus according to Claims 1 or 2, wherein in order to determine bilirubin in the blood, the reaction solution comprises 0.1 N hydrochloric acid and in addition to the capillary tube used for dispensing the blood sample, a second capillary tube is provided which is coated with 2,5-dichlorophenyl diazonium chloride.

5. An apparatus according to Claim 1 or 2, wherein for the determination of cholesterol in serum or plasma, the reaction solution com prises a potassium phosphate-buffered phenol methanol solution with the addition of hydroxy polyethoxy dodecane and in addition to the capillary tube used for dispensing the serum or plasma, a second capillary tube coated with 4-aminophenazone is provided for introducing a mixture of enzymes, consisting of cholesterol esterase, cholesterol oxidase and peroxidase.

6. An apparatus according to Claim 1 or 2, wherein for the determination of enzymatic blood sugar in a serum or plasma by the method according to Trinder, the reaction solution comprises phosphate-buffered phenol and in addition to the capillary tube used for dis pensing the serum or plasma sample, a second capillary tube is provided which is coated with a mixture of glucose oxidase, peroxidase and 4-aminophenazone.

7. An apparatus according to Claim 1 or 2, wherein for carrying out enzymatic blood sugar determination by the glucose dehydroge nase method, the reaction solution comprises a phosphate-buffer solution with a pH value 7.6 and in addition to the capillary tube used for dispensing the sample a second capillary tube is provided which is coated with a mixture of glucose dehydrogenase, mutrotase and nicotinamide adenine dinuclcotide (NAD).

8. An apparatus according to Claim 1 or 2, wherein for determining the choline esterase activity by the method according to Ellman, the reaction solution comprises a phosphate buffer solution with a pH value of 7.2 and in addition to the capillary tube used for dis pensing the sample a second capillary tube is provided which is coated with a mixture composed of acetyl thiocholine iodide and dithiobis-2-nitrobenzoic acid.

9. An apparatus for preparing a fluid for examination in optical analytic instruments, substantially as herein described with reference to the Examples.

10. An apparatus for preparing a fluid for examination in optical analytic instruments, substantially as herein described with reference to the accompanying drawing.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. [4] H. POPPER et al.: Biochem. Z. 291 (1937) 354 [5] P. RÖSCHLAU et al.: 9th Int. Congr. on Clin. Chemistry, Toronto, 1975 Abstr. No. 1 [6] P. TRINDER: Ann. Clin. Biochem. 6 (1969) 24 [7] D. BANAAUCH et al.: -Z. Klin, Chem. Klin, Biochem. 13(1976)101 [8] Gt. ELLMAN et al.: Biochem. Pharmacol. 7(1961) 88 [9] G. FRANK: Z. Analyt. Chem. 279 (1976) 155 WHAT WE CLAIM IS:

1. An apparatus for preparing a fluid for examination in optical analytic instruments comprising a sealed vessel containing an accurately prepared reaction solution and a capillary tube which is insertable into the vessel for dispensing a sample fluid into the reaction solution, wherein in order to regulate the optical properties of the fluid for examination at least one of the reactants required for the formation of the said fluid for examination from the said reaction solution is located in the solid phase in the said capillary tube used for dispensing the sample fluid and/or in a second capillary tube for introduction into the vessel.

7. An apparatus according to Claim 1 or 2, wherein for carrying out enzymatic blood sugar determination by the glucose dehydroge nase method, the reaction solution comprises a phosphate-buffer solution with a pH value

7.6 and in addition to the capillary tube used for dispensing the sample a second capillary tube is provided which is coated with a mixture of glucose dehydrogenase, mutrotase and nicotinamide adenine dinuclcotide (NAD).