AU491237B2 - Blood control standard - Google Patents

Description

69,958 174 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 com-

ponents 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 develope

for conveniently analyzing multiple components of blood serum. Illustrat

of the analytical equipment for these purposes are the Technicon "Auto-

Analyzer", the Warner Chillcott "Robot Chemist", the Beckman "Discrel

Analyzer" and the Hycel "Mark 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 moi

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, particulai

in the case of multi-automated procedures, are dependent upon rigid and

constant standardization of the biochemical determinations.

69,958 174 are standards used in actual practice Illustrative of the control ammonium is reconstituted with aqueous the freeze-dried serum which 466, 249, and described in U. S. Patent 3, bicarbonate prior to use as recon- standard which does not require the liquid blood serum control in U. S. Patent 3, 682, 835. stitution prior to use as disclosed other such used in making these and A principal raw material donor blood plasma obtained from blood blood control standards is stored collected and stored Blood plasma is normally centers and blood banks. citrate, such as, for example, sodium in various anticoagulant materials used tetraacetate. Certain widely heparin and sodium ethylenediamine additionally, dextrose (D-glucose). anticoagulant materials contain, is a acid, sodium citrate and dextrose) ACD blood (containing citric containing elevated anticoagulant stored blood principal example of an blood is CPD blood such anticoagulant stored levels of dextrose. Another

and dextrose). (containing citrate, phosphate of anticoagulant materials containing Due to the extraneous addition product will. or blood plasma, the stored dextrose to the stored blood

high, level of dextrose. Consequently, contain an elevated, or abnormally use as a raw material is not generally suitable for such stored blood plasma the case of so-called control standards except in in the preparation of blood desired. high levels of dextrose are "abnormal" control sera where a of the present invention to provide Accordingly, it is an object containing elevated anticoagulant stored plasma blood control standard from

levels of dextrose.

491.237

69.958 ri7

It is another object of this invention to provide a

method for preparing a blood control standard having normal

or red'uced 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 dis-

closure hereof.

Accordingly, there is provided by the present invention

a method of making a blood control standard from anticoagul-

ant stored blood plasma containing abnormally high levels of

dextrose comprising selectively destroying dextrose in said

blood plasma by aerobic fermentation with yeast in the

negative acceleration phase or in the stationary phase of

the yeast growth and separating particulate residue from the

fermentation broth,

491.237

69, 958 74

Many microorganisms, including yeasts, are capable of dividing

at a rapid rate, e. 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 little or no of a new medium with spores or with an old culture, a period

This is referred to as the lag phase. Eventually, exponential growth occurs.

multiplication begins. After a period of time, the multiplication slows down

late log phase. in what is termed a period of negative acceleration or the

of new cells in Finally, the death of cells balances or exceeds the formation

what is termed the stationary phase. The stationary phase includes the so-

called "real" stationary phase and the phase of decline in growth.

69 958 174

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 regulate temperati by providing suitable conditions of nutrient, oxygen supply, pH,

and inoculant.

A further description of the kinetics of yeast growth and the foregc edite( growth phases are found in "The Chemistry and Biology of Yeasts, New York, 1958, at pages 252 21 by A. H. Cook, Academic Press Inc.,

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 (aerobi

and fermentation) the yeast produces completely oxidized products, water fern carbon dioxide, from dextrose, whereas the absence of air (anerobic

tation) results in oxidized products (carbon dioxide) in part and reduced

products (alcohol) in part.

In a preferred method of the invention, defibrinated plasma or blo

serum is incubated with the yeast for about 12 24 hours, preferably abo

18 hours, at normal room temperature (ca. 20-25o C).

Any available yeast can be used in accordance with this invention.

Yeasts are widely distributed, well-known microorganisms. Even as eai

as 1930, some 6000 cultures of yeast were available from the Centraalbu

voor Schimmelculturen, Baarn, Holland. The commercially important y

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

e. Torulopsis spherica, Torulopsis utilis (Candida utilis) and Candida

pseudotropicalis.

491,237

69958 174 available yeast products are Examples of suitable commercially Inc.,* and marketed by Standard Brands Fleischmfanfl's ActiveDry Yeast Corp. These marketed by Universal Foods Red Star Active Dry Yeast, Preparation compressed distiller's yeasts. yeast products are essentially known and grain alcohol production is well of these yeast products from e. the early patent of H. Fleischmalf, described in many patents, of the Vienna 1870), which is a modification U. S. No. 102, 387 (April 26,

Process. to the present invention can The amount of yeast used according 10 grams of from about 0. 1 gram to about vary somewhat. about one gram blood plasma is suitable and active dry yeast per liter of more per liter iskpreferred. mg. dextrose plasma may contain up to 500 Freshly stored.ACD blood may still be as of storage the dextrose level per 100 ml. and after 21 days invention, this In accordance with the present mreY high as 300 .mg. per 100 ml. precraol_ 0 50 mg. per 100 ml., andkpreferably dextrose level is reduced tokabe, without any This reduction is brought about to about 40 mg. per 100 ml. plasma. normal protein content of the substantial destruction of the the treated plasma is filtered Following the foregoing fermentation, or residue and the resulting filtrate or centrifuged to separate particulate standard of this use as the base blood control supernatant is retained for base control can then be added back to this invenition. Sufficient dextrose whereby predetermined dextrose level standard to provide any desired be prepared. For blood control standards can various normal or abnormal of from about can be increased to a range example, the dextrose level

to about 400 mg. per 100 ml.

49 1,2 37

69.95 '74 can be further treate The blood control standard prepared as above

sodium, potassium and to reduce the inorganic ion level, particularly as described in calcium, and/or to remove the lipoprotein components

U. S. Patent 3, 682,835. Thus, the above-prepared material can be admi CRz 3 isrrea Trace Ma, such as "Dowex-50"'to substantially with a strong cation exchange resin

can be removed by reduce said cation level, and the lipoprotein content

hydrocarbon. The trea extraction with a fat-solvent such as a chlorinated

water to about its original material is then conveniently reconstituted with out prior to the self volume. The foregoing treatment can also be carried

with yeast. destruction of the dextrose by the aerobic fermentation the invention althoug The following examples will further illustrate

examples. the invention is not limited to these specific

EXAMPLE 1

liters) was obtained f Pooled human ACD stored blood plasma (ten contain a blood donor center. Upon assay, the plasma was determined to

protein content of 345 mg. (mg. per 100 ml.) of glucose and had a total

with 30, 000 6. 5 gram The blood plasma was defibrinated by reaction

Co., Detroit, Michigan) of thrombin (Thrombin Topical, Parke, Davis

then removal of the clotted material. Upon assay, the defibrinated plasn

was determined to contain 350 mg. of glucose.

one gram of To one liter of the defibrinated plasma was added

Fleischmann's Active Dry Yeast. The mixture was stirred overnight (ab

(about 200 The 12 hours) on a magnetic mixer at room temperature matter and the filtral mixture was then filtered to remove the particulate

standard of the present was retained as the desired base blood control mg. of glucos, invention. Upon assay, it was determined to contain 10

491, 237

69,958 174 standard was mixed with The thus prepared base blood control

resin in 3 increments of 10 grams grams of "Dowex-50" ion exchange reduced from its original level of each whereby the Na+ ion level was

the K ion level was reduced from 163 meq. /liter to 97 meq. /liter and

to 4. 8 meq. /liter. The resin was its original level of 11 meq. /liter

increment by filtration through removed after treatment with each 10 gram

was determined to have a total glass wool. Upon assay, the final product

protein content of 6. 3 gram

EXAMPLE 2

prepared in Example 1, Another liter of the defibrinated plasma 3 "Dowex-50" ion exchange resin in above, was mixed with 30 grams of the Na+ ion level was reduced to increments of 10 grams each whereby

reduced to 4.4 meq. /liter. The resin 94 meq. /liter and the K+ ion was

each 10 gram increment by filtration was removed after treatment with was determined through glass wool. Upon assay, the resin-treated plasma

to contain 310 mg. of glucose.

mixed with one gram of The resin-treated plasma was then

stirred overnight (about 12 hours) on Fleischmann's Active Dry Yeast and

(about 200 The mixture was a magnetic mixer at room temperature matter and the filtrate was retained then filtered to remove the particulate

of the present invention. Upon as the desired base blood control standard

10 mg. of glucose and the total assay, it was determined to contain

protein content was 6. 4 gram

Claims (1)

1. 69,958 /74 The claims defining the invention are as follows:

   1. The method of making a blood control standard from

   anticoagulant stored blood plasma containing abnormally high

   levels of dextrose comprising selectively destroying dextrose

   in said blood plasma by aerobic fermentation with yeast in

   the negative acceleration phase or in the stationary phase of

   the yeast growth and separating particulate residue from the

   fermentation broth.

   2. The method of Claim 1 in which the yeast is Saccharomyce

   cerevisiae.

   3. The method of Claim I in which the blood plasma is

   defibrinated and then incubated with the yeast for 12 to 24

   hours at 200 to 25 °C

   4. The method of Claim 1 in which from 0.1 gram to 10 grams

   of yeast are used per litre of blood plasma.

   The method of Claim 1 in which the dextrose in said bloo

   plasma is reduced from an initial level of 300 to 500 mg. per

   100 ml. to a final level of 0 to 50 mg. per 100ml.

   6. The method of Claim 1 in which the yeast is Saccharomyce

   cerevisiae, the blood/is defibrinated and then incubated with

   from 0.1 gram to 10 grams of said yeast per litre of said blo 0 plasma for 12 to 24 hours at 200 to 25 C, and in which the

   dextrose in said blood plasma is reduced from an initial leve

   of 300 to 500 mg. per 100 ml. to a final level of 0 to 50 mg.

   per 100 ml.

   7. The method of making a blood control standard according

   to Claim 1, substantially as herein described with reference

   491,2 37

   69.958 p-4 to any one of the foregoing examples thereof.

   8. A blood control standard prepared by the method of any one

   of the preceding claims.

   DATED this 26th day of January, 1978.

   BAXTER TRAVENOL LABORATORIES, INC.

   By their Patent Attorneys,

   ARTHUR S. CAVE CO.