DE2156604A1 - Control standard for liquid blood serum - Google Patents

Description

PATENT LAWYERS

dr. W. Schalk dipl.-ing. P. Wirth dipl.-ing. G. Dannenberg DR. V. SCHMIED-KOWARZIK · DR. P. WE I N HOLD · DR. D. G U DEL

6 FRANKFURT AM MAIN GR. ESCHENHEIMER STRASSE 39

Baxter Laboratories, Inc. Morton Grave, 111.60053 United States

SK / SK H-233

Control standard for liquid blood serum

'■ · i

The present invention relates to a control standard sample for liquid blood serum and a method for producing the same.

Blood serum is a complex biological fluid with numerous components of essential physiological importance. With normal or average- ' For healthy people, the concentrations of these components fall within certain, reasonably well-defined limits. If it is determined that one or more of these components are analyzed outside of these acceptable limits are various diseases or pathological conditions of the body system displayed.

In recent years, various automated methods have been developed for convenient analysis of blood serum and its many components. Some analysis devices for these purposes is "Auto Analyzer" Technician of the company, the "Robot Chemist" of the company Warner Chillcott and the "Discrete Analyzer" Beckman. These instruments can be used quickly and sequentially. the concentration of a number of blood serum components in a single

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Determine the sample, i.e. e.g. up to a dozen or more values.

In performing the analytical tests on blood serum and other biological samples with the above and similar devices, Laboratoriuras standard materials or so-called "reference or control standards" must be used to calibrate and control the instrument. Accurate results in the use of these instruments, especially in the case of a multiple automated procedures, depend on strict and constant standardization of biochemical determinations.

An example of the control standards used in practice is the freeze-dried serum, which is mixed with aqueous ammonium bicarbonate before use is reconstituted (see U.S. Patent 3,466,249). For specific purposes however, a complete liquid control standard sample that did not require such reconstitution would be useful; in practice it could be one find widespread use.

It is therefore an object of the present invention to provide a liquid blood serum control standard sample.

Another goal is to create a stable blood serum reference standard, Bk- which can be used for direct calibration and control of multiple automated analysis processes.

According to the invention, liquid blood serum is now mixed with a strongly acidic cation exchange resin to reduce the sodium, potassium and calcium levels in the Serum treated, and the pH of the serum »irri by sufficiently alkaline Reagent that does not contain any sodium, potassium, calcium or ammonium ions, Retained or reinstated at approximately its original value. Then it will be the serum treated with resin and adjusted in its pH value dried :,

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and the lipoprotein component is extracted with a fat solvent. The serum, which is practically free of lipoprotein, is mixed with water reconstituted to give a liquid basic blood serum. Dependent on From the analysis of the starting material and the desired concentration of the Various components in the final product can cause this basic liquid blood flow Materials may be added to create the final control standard.

The method for the preparation of the liquid blood serum control standard according to the invention is particularly suitable for use with defibrinated blood plasma with high concentrations of inorganic ions: sodium, potassium and calcium such as stored blood plasma with the anticoagulants Sodium citrate, potassium oxalate, heparin sodium or sodium ethylenediamine tetra- " acetate, citrated blood plasma (the sodium citrate or sodium citrate and dextrose contains) ACD blood plasma (with citric acid, sodium citrate and dextrose) and CPD blood plasma (with citrate, phosphate and dextrose).

Defibrination is carried out by adding calcium ions, for example, to the citrated blood or "Polybrene" (trademark for hexadimethrene bromide) to heparinized Inflicts blood.

Examples of the strongly acidic cation exchange resins which can be used according to the invention are the sulfonic acid cation exchange resins. These resins may for example, like sulfonated phenol, sulfonated polystyrene, sulfonated phenol-formaldehyde, sulfonated copolymers of monoalkenyl aromatic hydrocarbons having from about 0.5-25 wt .-% of a crosslinking Dialkenylkomponente such Divinylbsnzöl, divinyltoluene, divinylxylene and similar resins to be. Such resins are under the trademarks "Dowex SO (Dow Chemical Co.)," Amberlite IR-120 "(Rohm & Haas Co., Inc.)," Driolite C-3 "(Chemical Process Co., Inc.) and

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"Diaion SK No. 1" (Mitsubishi Chemical Industries Ltd.) available from Hendel. A preferred resin is "Dowex 50"; This BS is a sulfonated one with divinylbenzene crosslinked polystyrene according to U.S. Patent 2,366,007.

Other strongly acidic cation exchange resins which can be used according to the invention are familiar to the person skilled in the art and can according to Kunin "Ion Exchange Resins", John Wiley & Sons, Inc., New York, 1950.

A preferred resin is an ion exchange resin based on polystyrene with nucleated sulfonic acid groups in the H 'state, such as "Dowex-50" with 50-100 mesh.

The blood serum is preferably in proportions of the ion exchange resin about 25-100 g resin p. .5 1 serum mixed. For example, about 40 g of "Dowex 50" to be mixed with 1 l of serum; thus excellent results are obtained, reducing the sodium ion level from a range of about 165-205 million equivalents per 1 serum reduced to about 90-95 milliequivalents per 1 serum will.

A preferred alkaline reagent to keep the serum pH around its To keep the original value or to adjust it to this is a tris- Buffer / jri- (hydroxymethyl) -aminomethany. The Tris buffer is preferably one aqueous solution of about 30-50 g Tris compound per 1 dist. Water and has a pH of about 5.8-6.8 before drying.

According to one embodiment of the present invention, an important im Control standard to contain blood coraponents of urea nitrogen or Blood urea nitrogen (BUN). The blood urea nitrogen can preferably according to the Bertholet test or a modified Bertholet test according to the US patents 3 119 751, 3 467 499 and Re-issue 26 125

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to be determined. However, it has been found that the use of Trips Buffer interferes with the determination of blood urea nitrogen according to the Bertholet or modified Bertholet test. Surprisingly, the use of. Lithium hydroxide or strontium hydroxide do not pass these BUN tests, whereby at the same time the advantage is achieved that the Tris buffer is free of potassium, sodium,

is. .
Calcium and ammonium ions / The lithium ion preferably used according to the invention

hydroxyd is an aqueous solution of about 5Q-7Q gj lithium hydroxide per 1 dist.

Water.

The drying of the serum treated with resin and adjusted in its pH value is preferably carried out by conventional freeze-drying or Lyophilisie- g approximation methods in which the serum from the frozen state under high vacuum dried and rapidly sublimated ice or other frozen solvent to give a porous solid is obtained.

Then the lipoprotein content of the dried serum was removed by extraction with a fat solvent. Fat solvents which can be used according to the invention are organic solvents such as esters, ethers, ketones, hydrocarbons and chlorinated hydrocarbons. The organic solvents are, for example hydrocarbons such as light paraffinic petroleum fractions or naphthas, particularly from hexane or heptane type, cyclic hydrocarbons such as ä \ cyclohexane, chlorinated solvents such as chloroform and carbon tetrachloride, ethers such as diethyl ether, and ketones such as acetone and Butanone. A preferred fat solvent is a fluorocarbon or "freon" such as monofluorotrichloromethane, dichlorodifluoromethane, monochlorodifluorraethane, monofluorodichloromethane, and the various mixtures thereof.

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Solvent extraction is done by thoroughly mixing the getrocknacsn Serum with the fat solvent and subsequent separation of the undissolved Materials from the fat solvent. The dried serum is mixed with the solvent mixed preferably in proportions of about 30-90 g serum per liter of solvent for about 10-60 minutes with stirring. The fat solvent can be filtered off or removed by similar procedures, with any remaining solvent from the resulting dry serum particles is allowed to evaporate.

The serum, which is practically free of lipoprotein, is then mixed with water, preferably least. Water, to about its original volume odBr in reconstituted in other appropriate proportions, thereby setting the standard of control obtained from liquid basic blood serum.

Blood serum components that are incorporated into the Kantroll standard according to the invention are substances from the group of albumin, acid phosphate- tase, alkaline phosphatase, amylase, bilirubin, calcium, carbon dioxide, Chloride, cholesterol, creatinine, glucose, hemoglobin, lactic acid dehydrogenase, Phosphorus, potassium, sodium, total protein, transaminases such as seruraglutamine oxaloacetic acid transaminase and serum glutamine pyruvic transaminase, triglycerides, urea nitrogen, uric acid and other such substances of the blood serum.

A Kantroll standard preferred according to the invention contains predetermined concentrations of the following blood serum components in a mixture: sodium, potassium, chloride, phosphorus, calcium, BUN, glucose, uric acid, creatinine, seed protein, protein-bound iodine (PBlJ and carbon dioxide (HCO ₃ "). The liquid" basic serum can be brought to the desired, predetermined values of these components by adding suitable amounts of the corresponding, the Korapo—

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nenten-containing materials adds. For the 12 above blood serum components the following materials can be added: sodium acetate, potassium hydroxide, sodium chloride, potassium dihydrogen phosphate, calcium chloride, Urea, glucose, uric acid, creatinine, dried blood serum, thyroxine and Sodium bicarbonate. This control standard is below normal refrigeration temperatures (4 C.) Storage stability for 3 months and provides suitable determination values for the components mentioned.

The following examples illustrate the present invention without it to restrict.

Example 1

Citrated human blood serum was made by adding calcium ions and an- " final filtration defibrinated. Then, the obtained serum was coated with a polystyrene ion exchange resin having kerigebundsnen sulfonic acid groups ("Dowex 50" j 50-100 mesh) mixed in a ratio of 40 g resin per 1 serum, the value of the sodium and potassium ions (and any CaI

In this scale,

cium ions) in the serum. / As the hydrogen ions are released into the serum by exchanging sodium and potassium ions, the pH of the serum is kept at a value of about 6.2 + 0.2 with an aqueous solution of lithium hydroxide or discontinued. The value of the sodium ions by this ion-exchange of about 165-205 milli-equivalents per 1 serum ä to about 90-95 milliequivalents per 1 serum reduced. The pH of the final treated serum is examined and adjusted to 6.2 + 0.2 with lithium hydroxide. The serum treated with resin and adjusted to its pH value is lyophilized and then extracted with "Freon TF" (trichlorotrifluoroethane) by thoroughly mixing 75 g of the dried serum with 1500 ecm "Freon". The mixture was filtered through Whatman No. 1 filter paper

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and spread the remaining solid to dry. The dry material was then with dist. Water and reconstituted to remove any remaining finely divided material filtered. This product was used as the control standard of the liquid basic blood serum and adjusted as desired Components examined.

In this example, 3 control standards were pre-determined with the following low, medium and high concentrations produced:

Component Control standard concentration *

Sodium (milliequiv. / L) Potassium (milliequiv. / L) Calcium (mg ³ /.) Chloride (milliequiv. / L) BUN (mg ^) Creatinine (mg ^ d) Uric acid (mg ⁰ ^) Glucose (mg ° / >) Total protein (gtrf / o) Albumin (gn $>) CO ₂ (HCO ₃ "") PBI (.

P (mg / o)

* each concentration is within + 10% of the specified value

To produce these three concentration strengths, the above was produced; practically lipoprotein-free dried serum divided into three parts and with crsi different amounts of dist. Reconstituted water, given below Protein values were used as a basis. The reconstituted = :! Samples showed the following analysis:

low conc. medium conc. high conc. Number 1 . No. 2 No. 3 125 140 155 3 5 7th 6th 10 14th 80 100 120 10 40 80 1.0 4.0 7.0 3.5 7.5 11.5 80 160 400 4.5 , 6.0 7.2 2.7 3.6 , 43 18th 25th 35 4.3 6.0 9.0 3 8th 13th

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component reconstitutes sample percent No. 2 ration '~ - number 1 96.9 No. 3 Sodium (milliequiv /./ l) 72.8 1.52 102 Potassium (milliequiv /./ l) 1.14 5.23 1.6 Calcium ^ iicßd) 3.93 95.95 5.5 Chloride (milliequiV. / L) 72.11 9.5 101 BUN (mg ⁰ /.) 7.14 0.76 10 Creatinine (mg ^ d) 0.57 2.7 0.8 Uric acid (mg) ^) 2.14 81.7 3 Glucose (rnc / jd) 61.4 6.0 86 Total protein (gtif / d) 4.5 3.6 6.3 Albumin (gm / o) 2.7 2.35 3.8 C0 _D (HCO ") 1.76 4.09 2.47 PBI (/ "£ $) 3.07 3.23 4.3 P (mg%) 2.43 3.4

The reconstituted samples 1, 2 and 3 were made by adding appropriate amounts the appropriate following materials to prepare the above control standards used in low, medium and high concentration

added material

added Me ng to e sample

number 1 No. 2 No. 3 Sodium acetate; G 2.38 1.53 0.467 Potassium hydroxide; ecm 1.01 1.16 1,382 Calcium chloride; G 3.45 7.97 0.142 Sodium chloride j g 0.239 ü, 058 0.513 Urea; G 0.034 0.366 0.84 Creatinine; mg 2.58 19.44 37.2 Uric acid; G 0.008 0.029 0.051 Glucose; G 0.112 0.470 ' 1,884 dried serum; G 0 0 9 Sodium bicarbonate; mg 818.5 1141.6 1639.5 Thyroxine; yul 20.2 31.3 77.05 Potassium dihydrogen phosphate; G 1,500 0.126 0.253

0 2 3 / I U 9 p

The adjusted control standard material was sterilized by a "Millipore" Filter (0.22 micron) filtered and used as final control standards given in ampoules; the standards are available to clinical laboratories and other such analytical and test laboratories which do such Need blood serum control standards.

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Claims (8)

Claims 1.- Procedure for the preparation of a liquid blood serum control standard blood plasma stored with anticoagulant, characterized in that that you can thoroughly clean the defibrinated plasma with a strongly acidic cation exchange resin mixes to the value of sodium, potassium and / or calcium ions to reduce significantly in the serum, the serum then dries that Lipoprotein components removed by extraction with a fat solvent and reconstitute the serum with water. 2.- The method according to claim 1, characterized in that the Kationenaus- « exchange resin with the defibrinated plasma in proportions of about 25-100 g Resin per 1 plasma mixes sufficient to keep the sodium level from about 165-205 " Milliequivalents per 1 serum to approximately 90-95 milliequivalents per 1 serum to lower. 3, - Method according to claim 1 and 2, characterized in that the dried Serum in proportions of about 30-90 g serum per 1 solvent with the fat solvent is mixed. 4. Process according to claims 1 to 3, characterized in that a chlorinated hydrocarbon, preferably trichlorotrifluoroethane, is used as the fat solvent is used. 5.- The method according to claim 1 to 4, characterized in that the pH of the resin-treated serum using a reagent from the group of aqueous Lithium hydroxide, strontium hydroxide or tris (hydroxymethyl) aminomethane set or held at a value of about 5.8-6.8 will. 209 823/109 5 6, - Method according to claim 1 to 5, characterized in that as ion exchange resin one based on polystyrene with core-bonded sulfonic acid groups in the H 'state is used. 7.- The method according to claim 1 to 6, characterized in that such an ion exchange resin based on polystyrene with core-bound Sulfanic acid groups used in the H 'state and with the defibrinated Plasca. is mixed in proportions of about 25-100 g resin per 1 plasma, as a fat solvent a chlorinated hydrocarbon is used and the dried one is used Serum mixed in proportions of about 30-90 g resin per 1 solvent and the pH of the serum treated with resin by means of a reagent from the group of lithium hydroxide, strontrium hydroxide or tris (hydroxymethyl) aminomethane is set or held at about 5.8-6.8. 8.- Liquid blood serum control standard, consisting of one with an anti- coagulant stored blood plasma, which defibrinates, to the essential Decrease in sodium, potassium and / or calcium ions with a strong treated with acidic cation exchange resin, dried, freed from the lipoprotein component by extraction with a fat solvent and then as a serum is reconstituted with water. The patent attorney: 209823/1095