DE2430622A1 - BLOOD CONTROL STANDARD AND METHOD FOR MANUFACTURING IT - Google Patents

Description

• · Ii III

PATENT LAWYERS

DipL-lng. P. WIRTH Dr. V. SCH MI ED-KOWARZIK Dipl.-Ing. G. DANNENBERG Dr. P. WEINHOLD Dr. D. GUDEL

β FRANKFURT AM MA ₁ M 2870M GB. ESCHENHEIMER STRASSE 3β

SK / SK

Case H-395; S3M: gk

Baxter Laboratories, Inc. Morton Grove, 111.60055 / USA

Blood control standard and procedure for its

Manufacturing

The present invention relates to a blood control standard and a process for its manufacture.

Blood serum is a complex biological fluid that has numerous Contains components of essential physiological importance. In normal or average healthy people the concentrations of these components fall in certain, fairly well-defined limits. If analysis determines that one or more of these components is outside of them various diseases or pathological conditions of the body are indicated.

In recent years, various automated methods have been used to easily analyze the various components of blood serum developed u / order. An analysis device for these purposes For example, the "Auto-Analyzer" from Techniconj is the "Robot

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Chemist "from Wanrer Chillcott, the" Discrete Analyzer "from Beckman and the "Mark X" from Hycel. These instruments can quickly and sequentially change the concentrations of a wide variety determine blood serum components in a single sample, e.g. up to a dozen or more values.

When performing analytical tests on blood serum and other biological samples with the above and similar devices, laboratory standard materials or so-called "reference or control standards" for calibrating and checking the instrument uses tuerden. The exact results of the l / er

Turn of these tools, especially in the case of multi-automated ones l / experienced, depend on the constant and strict standardization of the biochemical determinations.

Examples of the control standards used in practice are the freeze-dried sera that were prepared before the Use to be reconstituted with aqueous ammonium bicarbonate (see e.g. U.S. Patent 3,466,249) and the liquid blood serum control standard, which does not require reconstitution prior to use (see U.S. Patent 3,6B2,835).

One in the preparation of these and other such blood control standards The main raw material used is stored blood plasma from blood donation centers and blood banks. Blood plasma will usually in various ahticoagulant materials, uj'ie iiatrium citrate, heparin and iJsodium ethylenediaminetetraacetate, collected and stored. Certain commonly used anticoagulants also contain dextrose (D-glucose). ACD blood (da3 that contains citric acid, sodium citrate and dextrose) is a

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Main example of a "stored with an anticoagulant" Blood that contains high levels of dextrose. Another, such, Blood stored with anticoagulants is CPD blood (which contains citrate, phosphate and dextrose).

Due to the external addition of the dextrose-containing anticoagulant materials to stored blood or blood plasma, the stored blood contains an increased or abnormally high level of dextrose. Therefore, such a mounted plasma is usually not suitable for use .as · raw material in the production of blood control standards unless called irn case. "Abnormal" Kontrollsore.n _s are desired in high walchen Dextrosewerte.

The aim of the present invention is therefore to provide a blood control standard from plasma stored with anticoagulant containing elevated levels of dextrose.

Another aim is to create a method of manufacture a blood control standard with normal or decreased Dextrose extract from stored containing elevated dextrose values Plasma without adversely affecting normal protein constituents in this blood plasma.

Generally speaking, the objectives of the present invention are by selective destruction of the dextrose in the stored blood aerobic fermentation with yeast in the negative acceleration phase or reached in the stationary phase of yeast growth. It is important according to the invention to use these growth phases, since otherwise the K & fu from the blood proteins for the production of further

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(grows)
Yeast cells are alive / and the protein content of the product is undesirable

reduced u / ird.

Many microorganisms, including yeasts, can move at high speed, e.g. at a frequency of at least once an hour, if kept in favorable growing conditions. This leads to an exponential or logarithmic multiplication of the cells. Usually after the initial inoculation of a new medium with spores or with an old culture, a period with little or no Growth. This is usually called the lag phase designated. Gradually, the exponential increase then begins. After a certain time, the increase slows down, what is called the period of negative acceleration or the late log phase. After all, death equals of cells stops or exceeds the formation of new cells *? what is called the stationary phase. The stationary phase includes the so-called "real" stationary phase and the decrease phase, of growth.

Thus, in the logarithmic phase, the inches and parts grow while in the stationary phase they show little or no growth and division.

These different phases of yeast growth can be achieved through appropriate Conditions of nutrients, oxygen supply, pH value, Temperature and inoculants are regulated or controlled.

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Another description of the kinetic conditions of yeast growth and the above growth phases can be found in "The • Chemistry and Biology of Yeasts" by A.H. Cook, Academic Press Inc., New /, York, (1958), pages 252-275, which are hereby included in the tior » lying registration is also included.

In accordance with the invention, it is also important to be aerobic and not aerobic apply anaerobic fermentation. In the presence of air (aerobic fermentation) the yeast gives completely oxidized products, Water and carbon dioxide from dextrose, during the absence

from air (anaerobic fermentation) to partially oxidized pro-partially
products (carbon dioxide) and / reduced products (alcohol).

In a preferred method according to the invention defibrinated plasma or blood serum is about 12-24 hours, preferably about 18 hours (about 20-25 ⁰ C.) incubated at ordinary room temperature with the yeast.

Any available yeast can be used in accordance with the invention. Yeasts are widespread, well-known micro-organisms. Even Already in 1930 there were about 6000 yeast cultures from the central office voor Schimmelculturen, Baarn, Holland, available. the are commercially important courtyards that can be used according to the invention e.g. ' Saccharomyces cerevisiae, S. cerevisiae var. Ellipsoideüs, S. carlsbergensis, S. fragilis and the Torula Hefan, e.g. Torulopsia spherica, T. utilis (Candida utilis) and Candida -psaudotropicalis.

Examples of suitable, commercially available yeast products are Fleischmann's active dry yeast from Standard Brands

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Inc., and "Rod Star" active dry yeast8 from Universal Foods Corp. These yeast products are essentially compressed distillery yeasts. The manufacture of these yeast products from the Alcohol production from grain is known and described in many patents (see e.g. the early ÜS patent 102 387 (April 26, 1B7G), which is a modification of the Wiener Procedure is.)

The amount of yeast used according to the invention can vary somewhat. Usually between about 0.1-10 g of active dry yeast G per 1 blood plasma is used, about 1 g / l is preferred.

Freshly stored ACD blood plasma can contain up to 500 mg of dextrose included per 100 ecm; and after 21 days of storage the Dextrose value will still be 300 mg per 100 ecm. According to the invention this dextrose value is about 0-50 mg per 100 ecm, preferably about AO mg per 100 ecm, reduced. This reduction takes place without significant destruction of the normal protein content of the plasma.

After the above fermentation, the treated plasma is filtered or centrifuged to separate a finely divided residue, and the resulting filtrate or supernatant liquid is retained for use as the basic blood control standard of the present invention. Sufficient dextrose can then be re-added to this baseline control standard to establish any desired, predetermined level of dextrose, whereby various normal or abnormal blood control standards can be produced. For example, the dextrose value can be. can be increased to a range of atu / a 80-400 mg per 100 ecm .

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The blood control standard prepared above can be used further for Reduction of the fourth in inorganic ions, especially sodium, potassium and calcium, and / or to remove the lipoprotein components (see U.S. Patent 3,602,835). The material produced as above can be: strong cation exchange resin, such as "Dou / ex 50", is essential Reduce the cation value 'mixed, and the lipoprotein content can be removed by extraction with a fat solvent such as a chlorinated hydrocarbon. The treated material is then appropriately treated with water to about £ 5i3 reconstituted to original volume. The above treatment ' can also prevent the selective destruction of the dextrose by the aerobic fermentation can be carried out with yeast.

The following examples illustrate the present invention, without restricting them.

U ο is ρ i ο 1 1

10 l of combined tinnally light stored ACD blood plasma were obtained from a blood donation center. According to analysis, the plase contained 345 mg % (mg per 100 ecm) glucose and had a cscai-titanium protein content of 6.5 g %. The blood plasma was defibrinated by reaction with 30,000 units of thrpmbin ("Thrombin Topical" from Firna Parke, Davis & Co., Detroit, Michigan), whereupon the coagulated f-'material was removed. According to analysis, (las dbfibrinicrte plasma contained 350 rug % glucose.

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1 g of Fleischmann active dry yeast was added to 1 liter of the defibrinated plasma. The mixture was (2O ⁰ C. approximately) overnight (about 12 hours) with a magnetic mixer at room temperature, then filtered to remove finely divided material and retained as the filtrate according to the invention, desired Gundblutkontrollstandard. According to analysis, this contained 10 mg % glucose.

• The thus prepared Grundblutkontrolletandard ex-50 "ion exchange resin in three portions was mixed g of 10 with 30 g of ¹¹ Dom, whereby the Na ⁺ ion content from its original value of 16.3 meq / l 97 meq / l and the K ion content of his The original value was reduced from 11 meq / l to 4.8 meq / l. After treatment with 10 g portions each, the resin was removed by filtering through glass wool. According to analysis, the end product contained a total protein content of 6.3 g %.

Example 2

Another liter of the dBfibrinated Plaemaa prepared in Example 1 was mixed with 30 g of "Dowex-50" ion exchange resin in three portions of 10 g each, bringing the Na ⁺ ion value to 94 meq / l and the K ⁺ ion value to 4.4 meq / l has been reduced.

The resin was removed after treatment with 10 g portions by filtering through glass wool. The resin-treated plasma was analyzed to contain 310 mg % glucose.

Then the resin-treated plasma was mixed with 1 g of Fleiechmann's active dry yeast and stirred overnight (about 12 hours) at room temperature (about 20 ° C.) in a magnetic mixer. The mixture was then filtered to remove the finely divided material and the filtrate retained as the desired basal blood control standard of the present invention. According to analysis, it contained 10 mg % glucose and a total protein content

of 6.4 g %.

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Claims (6)

Pa t e η t a η s ρ γ ü c h a 1.j- Procedure for preparing a blood control standard a stored blood plasma containing anticoagulants with increased dextrose eggs, characterized in that one the dextrose in the blood plasma through aerobic fermentation with yeast in the negative acceleration phase or in the stationary phase of yeast growth selectively destroyed. 2. ~ The method according to claim 1, characterized in that as Yeast Saccharomyces cerevisiae is used. 3.- The method according to claim 1 and 2, characterized in that that the blood plasma is defibrinated and then with the yeast about 12-24 Hours at about 20-25 ° C. "Incubated and / or. - "4.- The method according to claim 1 to 3, characterized in that about 0.1-10 g of yeast per 1 blood plasma can be used. 5. Method according to claims 1 to 4, characterized in that the dextrose in the blood plasma is reduced from an initial value of about 300-500 mg per 100 ecm to a final value of about 0-50 mg per 100 ecm. 6.- The method according to claim 1, characterized in that as Yeast Saccharomycea cerevisiae used, the blood plasma is defibrinated UNOE then with about 0.1-10 g of yeast per 1 blood plasma about 12-2Ä hours bsi about 20-25 ⁰ C. is incubated, wherein the Dsxtroae in the blood plasma of an initial Worth of fltuia. 300-500 mg per 100 ecm to a final value of around. 0-50 mg per 100 ecm is reduced. 509809/0684 Blood control standard, consisting of a blood plasma stored with an anticoagulant with etu / a 300-500
mg "per 100 ecm of dextrose that is defibrinated and then with yeast by aerobic fermentation with 0.1-10 g of yeast per 1 plasma in the negative acceleration phase or in the stationary phase of the yeast / growth to selectively destroy the dextrose and reduce its concentration a value of about 0-50 mg per 100 ecm
is implemented murder. The patent attorney: (t 509809/0684