Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2496/00—Reference solutions for assays of biological material
  • G01N2496/05—Reference solutions for assays of biological material containing blood cells or plasma
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

• This invention relates to the preparation of clinical chemistry controls and standards.
• it relates to the manufacture of controls and standards having cholesterol and triglyceride concentrations above the normal mean for human serum.
• Clinical chemistry assays for lipids such- as cholesterol and triglycerides in serum are commonly conducted in the laboratory. The accuracy and precision of these assays are continuously evaluated by conducting the test sample assays in tandem with controls or calibrators.
• the controls and cali ⁇ brators contain a known amount of lipid and thus provide results against which the continuing performance of the assays can be compared.
• quantitative assays depend upon the preparation of a standard curve. Such curves are prepared by assaying a plurality of standards different predetermined quantities of the various lipids.
• controls, standards and calibrators will be col ⁇ lectively referred to as quality control materials.
• Lipid-containing control materials * for the assay of human test samples are designed to mimic the performance of the test samples as closely as possible.
• cholesterol and triglyceride supplements are added to a base plasma or serum they are supplied as complexes with protein, just as they are found in test samples.
• lipoproteins are available from a wide variety of sources such as plasma or eggs, and they have the additional advantage of water solubility.
• lipoproteins as carrier-solubil- izers for cholesterol or triglycerides
• fragility of the protein-lipid complexes. They are sensitive to the pH, ionic strength and temperature of their environment and to abuse such as shear and concentration effects.
• One particular problem has been the reduction in lipoprotein water solubility brought about by lyophilization of plasma or serum containing quality control materials. This reduction in solubility is evidenced by the development of turbidity in the materials. This is obviously unsatisfactory because the precipitated lipids will perform differently in lipid assays when compared with test
• U.S. Patents 4,045,176 and 3,955,925 disclose two types o quality control materials which are optically clear upon reconstitution and which contain normal or elevated concentra ⁇ tions of lipids.
• One control material was made by adding high density human lipoproteins (alpha lipoproteins) to substan ⁇ tially human serum. The high density lipoproteins were stable
• a second control material was made by adding isolated, non-primate lipoproteins to substantially human serum. Accord ⁇ ing to the patentees it was not necessary to first separate the high and low density lipoproteins as was required when using human lipoproteins, and that the low density non-primate lipo ⁇ proteins could be included without substantial adverse effect. Bovine lipoproteins were most preferred.
• U.S. Patents 4,045,176 and 3,955,925 teach that isolated bovine lipoprotein can be added to human blood serum without adverse effect, the same could not be said for the addition of isolated bovine lipoprotein to serum other than that from humans.
• U.S. Patent 3,764,556 teaches isolating cholesterol-rich fractions from egg or human plasma and adding them to bovine or equine sera. This was done to enhance the normally low triglyceride and cholesterol concentrations in bovine and equine sera, thereby.enabling their use as normal o elevated lipid controls in the assay of human serum test sam- ples.
• an animal serum or plasma-based lipid quality control material having cholesterol and triglyceride concentrations of at least the levels found in normal human plasma or serum.
• a stable quality control material can be prepared by combining a lipoprotein concentrate from animals with an animal sera if the lipoprotein concentrate is enriched in high density or alpha lipoproteins. Applicants have found that it is essential to remove animal chylomicrons as well as low den ⁇ sity and very low density lipoproteins from animal lipoprotein concentrates before supplementing animal serum with the concen ⁇ trate.
• the objects of this invention are achieved by mixing substantially normal animal plasma or serum with animal high density lipoproteins which are substantially free of animal chylomicrons and low density and very low density lipo ⁇ proteins.
• the resulting novel quality control materials shoul be substantially free of human plasma protein fractions. They can be reconstituted after lyophilization to yield clear solutions.
• the animal plasma or serum chosen as raw material for preparation of the lipoprotein concentrate is not critical, although for the purposes herein "animal” is intended to exclude all primates. Suitable sources are cows, horses, chickens, sheep and swine. While the alpha lipoprotein conten of animal plasma varies somewhat this should not be a major factor in selecting a suitable source other than from consider ⁇ ation of yield. Bovine and equine serum is most suitable, although bovine is preferred.
• the animal high density lipoproteins are obtained by known processes.
• the lipoprotein II fraction disclosed in Proksch et al. “Clin. Chem.” 22/8:1302-1305 (1976) is suit ⁇ able, as is the bovine high density lipoprotein fraction pro ⁇ quizzed in accordance with Jonas, "J. Biological Chemistry” 247(23) :7767-7772 (1972) or Example 1 of U.S. Patent 4,045,176.
• Animal lipoprotein concentrates useful herein will contain fr about 80% to 100% alpha lipoproteins by lipid electrophoresis (Sebia electrophoresis cell) and, if purified to a lipoprotein concentrate essentially free of other proteins, serum should have a specific gravity of greater than about 1.065. More preferably the lipoprotein content of the fractions will be about 90% to 95% alpha lipoproteins, preferably about 90%, wit the remainder being pre-beta and beta globulins. The less the amount of beta and pre-beta lipoproteins the more suitable the concentrate will be from the standpoint of clarity. However, very high alpha lipoprotein purity raises costs and increases the amount of concentrate needed to supply a given triglyceride value.
• Such concentrates will generally contain about from 0.30 - 0.50 g triglyceride/liter and about from 10 to 15 g cholesterol/liter. Cholesterol and cholesterol esters will constitute about from 20% to 45% by weight of the lipoprotein, and triglycerides about 15% to 30% by weight. Generally, one will select concentrates having the highest alpha lipoprotein concentration, followed by selection on the basis of the high ⁇ est cholesterol content.
• Non-lipoprotein serum proteins which are lyophilization stable, e.g., X ⁇ -globulins, can be included in the lipoprotein concentrates used herein. It is not desir ⁇ able to have large amounts of gamma globulin, fibrinogen or albumin in the concentrate.
• pooled animal serum will function as the base to which the concentrate is added. It is preferred to use serum from an animal having a low beta and pre-beta lipoprotein content when compared with human plasma. Suitable sources are bovine, equine and sheep serum, with bovine being preferred. Commercially available animal plasma is easily converted to serum by clotting with calcium and thrombin and removing the clot by centrifugation, along with any remaining contaminant blood cells. Serum is stored frozen. The serum is a clear, amber fluid from which cellular elements and most fibrinogen have been removed, and it contains components of .clinical interest in assays other than those for lipids, e.g., salts, enzymes, glucose, protein and urea.
• lipids e.g., salts, enzymes, glucose, protein and urea.
• the serum should be sup ⁇ plemented with any of these components which are clinically significant if it is normally deficient in them, as is conven ⁇ tional practice in the art.
• the cholesterol and triglyceride components are supplemented b adding the lipoprotein concentrate described above to the serum.
• the base serum may also be processed to reduce its bet and pre-beta lipoprotein content, e.g. by selective precipita ⁇ tion with heparin or dextran sulfate in accordance with known processes. All serum starting materials should be free of chylomicrons.
• the general process for making quality control materials ordinarily starts with thawing and pooling sera which had been prepared and stored previously.
• Pooling means combining the sera from a plurality of separate donors into batches that can be economically processed on a commercial scale.
• the pooled serum is filtered through successively finer filters, termin ⁇ ating in filtration through a 0.3u pore diameter filter.
• albumin, cholesterol and triglycerides are added to bring the serum up to the desired protein, cholesterol and triglycerides concentrations.
• the alpha lipoprotein concen- trates can provide all cholesterol which is needed and in most cases all the protein and triglycerides as well. If not, then protein and triglycerides can be supplemented from the exogen ⁇ ous sources. Any other constituents that are present in insufficient concentration are then added as required.
• the pooled, adjusted serum is filled into vials, lyophilized and sealed.
• the amount of concentrate to be added to the serum depend upon the amount of triglycerides and cholesterol present in the base serum and in the concentrate. These quantities can be measured conventionally and the proper quantity of concentrate to be added readily calculated. Since the concentrate is more suitable for cholesterol enhancement this will be the control ⁇ ling component. Generally, the high density lipoprotein con ⁇ centrate will contain about from 20% to 45% cholesterol by weight of lipoprotein, but this will vary depending upon the species from which the lipoprotein was obtained and the parti ⁇ cular lipoprotein fraction obtained by the concentration method employed in that particular instance.
• the concentrate is typically added to bring the base serum cholesterol content at least up to the level of normal human serum, i.e. about from 1.2 to 2.5 g cholesterol (free plus ester)/ liter.
• Triglyceride should be brought up to about from 0.3 to 0.5 g/liter if possible without introducing more cho ⁇ lesterol than is desired.
• This invention has its principal utility in making hypercholesterolemic sera, i.e., sera in which the cholesterol content is about from 2 ⁇ 5 to 4.5 g/liter. Sera having concentrations throughout this range may be lyophilized and reconstituted to optically satisfactory solutions.
• the pooled animal sera produced by combining the above- described concentrates with an animal serum base contain a con ⁇ centration of lipoprotein lipid in excess of that found in pooled serum from animals of the same species.
• the composi ⁇ tions of this invention include pooled animal sera, particu ⁇ larly that from cattle and horses, which contain lipoprotein- bound cholesterol at a cholesterol concentration of greater than about 1.2g/l, preferably greater than about 2.0 g/1 and up to about 4.5g/1.
• novel products of this invention contain an enriched proportion of alpha lipoproteins, i.e., a proportion by weight of total lipoproteins and by weight/volume above that found in normal pooled serum and frequently a level above that in the serum of hyper ⁇ holesterolemi ⁇ specimens of the animal in question. Frequently the alpha lipoprotein concentration will be in excess of 25% weight/volume above that of normal pooled plasma or serum.
• the level of alpha lipoprotein also may be described in terms of the weight ratio of alpha lipoproteins to beta and pre-beta lipoproteins. In the case of bovine sera, the ratio of alpha lipoproteins to beta and pre-beta lipopro ⁇ teins is at least 3.75:1, usually about 4:1.
• Sheep sera havin a ratio of at least 3.1 to 1, preferably about 3.5 to 1 are also useful but not as desirable as the more conveniently available bovine serum.
• the lipid, e.g. cholesterol concen ⁇ tration is generally at least about 25% by weight/volume above that of normal pooled plasma or serum. - li ⁇
• lt is contemplated that cholesterol concentrates from on animal species can be added to a base serum of another animal.
• bovine concentrates can be used with normal, pooled horse serum.
• the products herein are preferably substantially free of human lipoproteins, contaminant human lipoproteins may accompany supplemental analytes such as human proteins added to adjust analyte concentrations as described above.
• the former assay demonstrated only a faint band at the very low density and chylomicron positions, but had a strong, broad stain identified as high density lipoprotein shading faintly into low density lipoprotein.
• This concentrate was clarified by filtration through a 0.3u pore diameter filter to a final volume of 49,800 ml.

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• 1983
  • 1983-04-20 EP EP19830901778 patent/EP0105923A4/fr not_active Withdrawn
  • 1983-04-20 WO PCT/US1983/000571 patent/WO1983003680A1/fr not_active Application Discontinuation

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