Blood control standard

Abstract

A blood control standard is prepared from anticoagulant stored blood plasma containing elevated levels of dextrose by aerobic fermentation with yeast in the late log phase or in the stationery phase of the yeast growth to selectively destroy dextrose without adversely affecting the protein constituents of said blood plasma.

Description

This invention relates to a blood control standard and method of preparation thereof.

Blood serum is a complex biological fluid containing numerous components of substantial physiological importance. In the normal or average healthy person the concentrations of these components fall within certain reasonably well-defined limits. When one or more of these components is determined upon analysis to fall outside of these acceptable limits, various diseases or pathological conditions of the body systems are indicated.

In recent years various automated procedures have been developed for conveniently analyzing multiple components of blood serum. Illustrative of the analytical equipment for these purposes are the Technicon "Auto-Analyzer," the Warner Chillcott "Robot Chemist," the Beckman "Discrete Analyzer" and the Hycel "Mark X." These instruments are capable of rapidly and sequentially determining the concentrations of a host of blood serum components in a single sample, for example, up to a dozen or more values.

In the performance of the analytical tests made by the above and similar equipment on blood serum and other biological samples, it is necessary to use laboratory standard materials or so-called "reference or control standards" for purposes of calibration and control of the instrument. Accurate results in the use of these instruments, particularly in the case of multi-automated procedures, are dependent upon rigid and constant standardization of the biochemical determinations.

Illustrative of the control standards used in actual practice are the freeze-dried serum which is reconstituted with aqueous ammonium bicarbonate prior to use as described in U.S. Pat. No. 3,466,249, and the liquid blood serum control standard which does not require reconstitution prior to use as disclosed in U.S. Pat. No. 3,682,835.

A principal raw material used in making these and other such blood control standards is stored blood plasma obtained from blood donor centers and blood banks. Blood plasma is normally collected and stored in various anticoagulant materials such as, for example, sodium citrate, heparin and sodium ethylenediamine tetraacetate. Certain widely used anticoagulant materials contain, additionally, dextrose (D-glucose). ACD blood (containing citric acid, sodium citrate and dextrose) is a principal example of an anticoagulant stored blood containing elevated levels of dextrose. Another such anticoagulant stored blood is CPD blood (containing citrate, phosphate and dextrose).

Due to the extraneous additon of anticoagulant materials containing dextrose to the stored blood or blood plasma, the stored product will contain an elevated, or abnormally high, level of dextrose. Consequently, such stored blood plasma is not generally suitable for use as a raw material in the preparation of blood control standards except in the case of so-called "abnormal" control sera where high levels of dextrose are desired.

Accordingly, it is an object of the present invention to provide a blood control standard from anticoagulant stored plasma containing elevated levels of dextrose.

It is another object of this invention to provide a method for preparing a blood control standard having normal or reduced levels of dextrose from stored plasma containing elevated levels of dextrose without adversely affecting the normal protein constituents of said blood plasma.

These and other objects of the invention will be apparent to those skilled in the art after reading the disclosure hereof.

Briefly stated, the objects of the present invention are achieved by selective destruction of the dextrose in stored blood by aerobic fermentation with yeast in the negative acceleration phase or in the stationary phase of the yeast growth. It is important to use these growth phases in the practice of the present invention, otherwise the yeast feeds on the blood proteins to make more yeast cells and the protein content of the product is undesirably reduced.

Many microorganisms, including yeasts, are capable of dividing at a rapid rate, e.g., at a frequency of less than once per hour, if kept under favorable growth conditions. This leads to a multiplication of cells which is exponential or logarithmic. Ordinarily, following initial inoculation of a new medium with spores or with an old culture, a period of little or no growth occurs. This is referred to as the lag phase. Eventually, exponential multiplication begins. After a period of time, the multiplication slows down in what is termed a period of negative acceleration or the late log phase. Finally, the death of cells balances or exceeds the formation of new cells in what is termed the stationary phase. The stationary phase includes the so-called "real" stationary phase and the phase of decline in growth.

Thus, in the log phase, the cells are growing and dividing whereas in the stationary phase the cells exhibit little or no growth and division.

These various phases of yeast growth can be controlled or regulated by providing suitable conditions of nutrient, oxygen supply, pH, temperature and inoculant.

A further description of the kinetics of yeast growth and the foregoing growth phases are found in "The Chemistry and Biology of Yeasts," edited by A. H. Cook, Academic Press Inc., New York, 1958, at pages 252 - 275, which is incorporated herein by reference.

It is also important in the practice of the present invention to use aerobic rather than anerobic fermentation. In the presence of air (aerobic fermentation) the yeast produces completely oxidized products, water and carbon dioxide, from dextrose, whereas the absence of air (anerobic fermentation) results in oxidized products (carbon dioxide) in part and reduced products (alcohol) in part.

In a preferred method of the invention, defibrinated plasma or blood serum is incubated with the yeast for about 12 - 24 hours, preferably about 18 hours, at normal room temperature (ca. 20.degree.-25.degree. C).

Any available yeast can be used in accordance with this invention. Yeasts are widely distributed, well-known microorganisms. Even as early as 1930, some 6,000 cultures of yeast were available from the Centraalbureau voor Schimmelculturen, Baarn, Holland. The commercially important yeasts which can be used in the practice of this invention are those such as Saccharomyces cerevisiae, Saccharomyses cerevisiae var. ellipsoideus, Saccharomyces carlsbergensis, Saccharomyces fragilis, and the Torula yeasts, e.g., Torulopsis spherica, Torulopsis utilis (Candida utilis) and Candida pseudotropicalis.

Examples of suitable commercially available yeast products are Fleischmann's Active Dry Yeast marketed by Standard Brands Inc., and Red Star Active Dry Yeast, marketed by Universal Foods Corp. These yeast products are essentially compressed distiller's yeasts. Preparation of these yeast products from grain alcohol product is well known and described in many patents, e.g., the early patent of H. Fleischmann, U.S. Pat. No. 102,387 (Apr. 26, 1870), which is a modification of the Vienna Process.

The amount of yeast used according to the present invention can vary somewhat. In general, from about 0.1 gram to about 10 grams of active dry yeast per liter of blood plasma is suitable and about one gram per liter is preferred.

Freshly stored ACD blood plasma may contain up to 500 mg. dextrose per 100 ml. and after 21 days of storage the dextrose level may still be as high as 300 mg. per 100 ml. In accordance with the present invention, this dextrose level is reduced to about 0 - 50 mg. per 100 ml., and preferably to about 40 mg. per 100 ml. This reduction is brought about without any substantial destruction of the normal protein content of the plasma.

Following the foregoing fermentation, the treated plasma is filtered or centrifuged to separate particulate residue and the resulting filtrate or supernatant is retained for use as the base blood control standard of this invention. Sufficient dextrose can then be added back to this base control standard to provide any desired predetermined dextrose level whereby various normal or abnormal blood control standards can be prepared. For example, the dextrose level can be increased to a range of from about 80 to about 400 mg. per 100 ml.

The blood control standard prepared as above can be further treated to reduce the inorganic ion level, particularly sodium, potassium and calcium, and/or to remove the lipoprotein components as described in U.S. Pat. No. 3,682,835. Thus, the above-prepared material can be admixed with a strong cation exchange resin such as "Dowex-50" to substantially reduce said cation level, and the lipoprotein content can be removed by extraction with a fat-solvent such as a chlorinated hydrocarbon. The treated material is then conveniently reconstituted with water to about its original volume. The foregoing treatment can also be carried out prior to the selective destruction of the dextrose by the aerobic fermentation with yeast.

The following examples will further illustrate the invention although the invention is not limited to these specific examples.

EXAMPLE 1

Pooled human ACD stored blood plasma (10 liters) was obtained from a blood donor center. Upon assay, the plasma was determined to contain 345 mg. % (mg. per 100 ml.) of glucose and had a total protein content of 6.5 gram %. The blood plasma was defibrinated by reaction with 30,000 units of thrombin (Thrombin Topical, Parke, Davis & Co., Detroit, Michigan) and then removal of the clotted material. Upon assay, the defibrinated plasma was determined to contain 350 mg. % of glucose.

To one liter of the defibrinated plasma was added 1 gram of Fleischmann's Active Dry Yeast. The mixture was stirred overnight (about 12 hours) on a magnetic mixer at room temperature (about 20.degree. C). The mixture was then filtered to remove the particulate matter and the filtrate was retained as the desired base blood control standard of the present invention. Upon assay, it was determined to contain 10 mg. % of glucose.

The thus prepared base blood control standard was mixed with 30 grams of "Dowex-50" ion exchange resin in three increments of 10 grams each whereby the Na.sup.+ ion level was reduced from its original level of 163 meq./liter to 97 meq./liter and the K.sup.+ ion level was reduced from its original level of 11 meq./liter to 4.8 meq./liter. The resin was removed after treatment with each 10 gram increment by filtration through glass wool. Upon assay, the final product was determined to have a total protein content of 6.3 gram %.

EXAMPLE 2

Another liter of the defibrinated plasma prepared in Example 1, above, was mixed with 30 grams of "Dowex-50" ion exchange resin in three increments of 10 grams each whereby the Na.sup.+ ion level was reduced to 94 meq./liter and the K.sup.+ ion was reduced to 4.4 meq./liter. The resin was removed after treatment with each 10 gram increment by filtration through glass wool. Upon assay, the resin-treated plasma was determined to contain 310 mg. % of glucose.

The resin-treated plasma was then mixed with 1 gram of Fleischmann's Active Dry Yeast and stirred overnight (about 12 hours) on a magnetic mixer at room temperature (about 20.degree. C). The mixture was then filtered to remove the particulate matter and the filtrate was retained as the desired base blood control standard of the present invention. Upon assay, it was determined to contain 10 mg. % of glucose and the total protein content was 6.4 gram %.

Various other examples and modifications of the foregoing examples will be apparent to those skilled in the art after reading the foregoing specification and the appended claims without departing from the spirit and scope of the invention. All such further examples are included within the scope of the invention as defined by the following claims.

Claims

What is claimed is:

1. The method of making a blood control standard from anticoagulant stored, defibrinated blood plasma containing elevated levels of dextrose which comprises selectively destroying dextrose in said blood plasma by aerobic fermentation with agitation utilizing 0.1 to 10 grams of a yeast in the negative acceleration phase or in the stationary phase of the yeast growth per liter of said plasma and separating particulate residue from the fermentation product.

2. The method of claim 1 in which the yeast is Saccharomyces cerevisiae.

3. The method of claim 1 in which the blood plasma is incubated with the yeast for about 12 to about 24 hours at about 20.degree. to about 25.degree. C.

4. The method of claim 1 in which the dextrose in said blood plasma is reduced from an initial level of about 300 to 500 mg. per 100 ml. to a final level of about 0 to 50 mg. per 100 ml.

5. The method of claim 1 in which the yeast is Saccharomyces cerevisiae, the blood plasma is incubated with said yeast for about 12 to about 24 hours at about 20.degree. to about 25.degree. C, and in which the dextrose in said blood plasma is reduced from an initial level of about 300 to 500 mg. per 100 ml. to a final level of about 0 to 50 mg. per 100 ml.

6. A liquid blood control standard comprising anticoagulant-stored blood plasma containing from about 300 to 500 mg. per 100 ml. of dextrose which is defibrinated, reacted with yeast by aerobic fermentation with agitation utilizing from 0.1 to 10 grams of said yeast per liter of said plasma in the negative acceleration phase or in the stationary phase of the yeast growth to selectively destroy said dextrose and reduce its concentration to a level of about 0 to 50 mg. per 100 ml, and separated from particulate residue.