US 3733179 A
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P. METHOD AND APPARATUS FOR THE QUANTITATIVE DETERMINATION OF May 15, 1973 F. GUEHLER BLOOD CHEMICALS IN BLOOD DERIVATIVES Filed Aug. 29, 1968 P404 fi' GUEHLER H i;: x l 1: ix n /H I IW V LTWQ L 6 a a wax M United States Patent (3 3,733,179 METHOD AND APPARATUS FOR THE QUANTITA- TIV E DETERMINATION OF BLOOD CHEMICALS IN BLOOD DERIVATIVES Paul F. Guehler, White Bear Lake Township, Ramsey County, Minm, assignor to Minnesota Mining and Manufacturing Company, St. Paul, Minn.
Filed Aug. 29, 1968, Ser. No. 756,098 Int. Cl. B65d 79/00; G01n 31/02, 33/16 US. Cl. 23-230 B 4 Claims ABSTRACT OF THE DISCLOSURE A method for the quantitative determination of a blood chemical in a blood derivative wherein a first container containing a blood derivative sample and a solution capable of forming therewith a reactive component is joined by filter means to a second container of test material capable of reacting with the reactive component to form a colored solution having an optical density dependout upon the concentration of the blood chemical. The contents of the compartments are combined by centrifuging the assembly. The optical density of the colored fluid is measured and is correlated with the concentration of the blood chemical. Apparatus for practicing this method.
The present invention relates to a method and apparatus for the quantitative determination of diagnostically significant chemicals in blood derivatives.
Abnormally high or low concentrations of certain blood chemicals may signal the onset or presence of many body illnesses. For example, abnormally low concentrations of cholesterol may signal a defective intestinal absorption of fats, liver disease, and overfunction of the thyroid gland. Continued abnormally high concentrations of cholesterol may foretell coronary thrombosis and may denote hereditary defects in fat metabolism, under-function of the thyroid gland, diabetes, and jaundice due to bile duct obstruction. Abnormal concentration of uric acid in blood are indicative of incipient gout and kidney stones. Methods for the quantitative determination of cholesterol and other blood chemicals constitute important clinical tool for the diagnosis of many body illnesses.
Heretofore, quantitative determinations of blood chemicals have been awkward and expensive and have required numerous time-consuming manipulative steps by experienced technicians. For example, in the determination of blood cholesterol by the modified method of Henly et a1. (as reported in Henry, R. J., Clinical Chemistry: Principles and Techniques, Harper and Row, New York, 1965, pp. 855-857), a technician must first measure out and combine quantities of a blood derivative (e.g. blood serum) and a solution of ferric chloride and acetic acid in a test tube. The ingredients are mixed by agitation and are set aside for -15 minutes. The test tube is then heated in a water bath for two minutes, cooled, and centrifuged to separate out precipitated protein. Into a measured quantity of the supernate is then mixed a measured amount of concentrated sulfuric acid. After again waiting for 10 minutes, the optical density of the resulting solution is photometrically measured and is correlated with the concentration of cholesterol in the blood derivative.
In similar fashion, determinations of such blood chemicals as uric acid, bilirubin, protein, glucose and urea have required such manipulative steps as measuring numerous ingredients together, preparing standard solutions immediately before use, etc.
Because of the time and skill needed for the many manipulative steps heretofore required for blood chemical analysis such as those described above, such analysis 3,733,179 Patented May 15., 1973 have been limited primarily to hospital medical laboratories.
It is an object of the present invention to provide a method and apparatus for the rapid quantitative determination of cholesterol and other diagnostically significant chemicals in blood derivatives.
It is another object of the present invention to provide a method and apparatus for the quantitative determination of cholesterol and other diagnostically significant chemicals in a blood derivative which requires few manipulative steps and minimum operator proficiency and which is simple and inexpensive.
Briefly, the method of the present invention comprises the sequential steps of (1) providing a first container of a solution capable of extracting a blood chemical from a blood derivative to form a component capable of reacting with a test material to form a colored fluid having an optical density dependent upon the concentration of said blood chemical in said blood derivative;
(2) adding to said first container a sample of said blood derivative containing said blood chemical to form said component;
(3) providing a second container containing said test material;
(4) joining said containers With connector means having air-permeable filter means chemically resistant to and normally separating the solution of said first container and the material of said second container, said filter means when joined to one of said containers, being capable of preventing the passage therethrough of one of said component and said material under normal gravitational force and being capable of permitting passage therethrough of said one of said component and said material under a force substantially greater than normal force of gravity such as a force generated during centrifugation (e.g., up to 1500 times the normal force of gravity);
(5) centrifuging said joined containers to cause to combine the contents thereof and to form, with agitation, a colored fluid; and
(6) measuring the optical density of said colored fluid.
Apparatus for practicing the method of the present invention is illustrated in the accompanying drawing wherein:
FIG. 1 is a cross-sectional representation of the preferred apparatus of the present invention as viewed immediately prior to centrifugation;
FIG. 2 is a cross-section view of the apparatus of FIG. 1 taken along line 22 of FIG. 1; and
FIG. 3 is a perspective exploded View of the apparatus of FIG. 1.
As used herein, the term blood chemical refers to cholesterol and to such other diagnostically significant chemical ingredients of blood as, for example, uric acid, bilirubin, protein, glucose, urea, etc.
By virtue of the present invention, the reactants required for blood chemical analysis may be supplied in premeasured quantities in suitable containers to the laboratory technician who merely adds to one of the containers a sample of a blood derivative, joins the two containers together with the filter means, centrifuges the assembly, and measures the optical density of the resulting colored fluid, obviating the necessity of measuring and manually transferring the various reactants and thus reducing the human error associated with such manipulations.
The method and apparatus of the present invention are adapted to be used for blood chemicals, the determinations of which may be reduced to the process of (1) preparing a liquid which contains the blood chemical to be analyzed in reactive form; that is, in a form capable of reacting with a test material to form a colored fluid having an optical density dependent upon the concentration of the blood chemical;
(2) combining this liquid with the test material to form the colored fluid; and
(3) photometrically measuring the optical density of the colored fluid.
For example, the determination of total cholesterol by the procedure reported in Henry, supra, p. 855, may be carried out by the method of the present invention as follows:
A measured sample of blood serum containing cholesterol is added to a first compartment, e.g. a glass vial, containing a premeasured amount of ferric chloride/ acetic acid reagent. This reagent precipitates protein and extracts cholesterol and cholesterol esters from the serum. A connector member having filter means is attached to the first compartment and this assembly is then attached to a second container, e.g. a second glass vial, containing a premeasured quantity of concentrated sulfuric acid. The filter means permits the assembly to be inverted during attachment thereof to the second container, preventing escape of the contents of the first container and avoiding spillage of the sulfuric acid from the second container. Centrifugation causes the contents of the first container to filter into the second container, forming two layers which, when subsequently mixed by gentle agitation, form a colored fluid. The optical density of the colored fluid is then measured and is correlated with the concentration of total cholesterol by known algebraic or graphic methods.
If the concentration of only cholesterol esters in blood is desired, a blood derivative containing cholesterol solely in ester form is substituted for the blood sample in the above example. Such a blood derivative may be prepared, for example, by precipitating free cholesterol from a sample of blood serum in an ethanol ether solvent by addition of digitonin, evaporating the solution to dryness, extracting cholesterol esters from the residue with petroleum ether, evaporating the petroleum ether and dissolving the remaining cholesterol esters in acetic acid. This procedure is more fully described in Henry, supra, pp. 857-858.
Determinations of bilirubin in blood serum by a modification of the Malloy-Evelyn procedure [Malloy and Evelyn, J. Biol. Chem. 119, 481 (1937)] may be carried out by the method of the present invention by adding a measured sample of blood serum containing bilirubin to a first compartment containing a premeasured quantity of a methanol-water-hydrochloric acid solution, attaching thereto the connector member having filter means, attaching this assembly to a second container containing a diazonium salt capable of reacting with bilirubin to form colored azobilirubin, centrifuging to cause the contents of the first compartment to filter into the second compartment to cause formation therein of a colored azobilirubin solution, and correlating the optical density of the colored azobilirubin solution with the concentration of bilirubin in the blood serum sample by known means, as described above.
Similarly, the concentration of uric acid in blood serum may be determined by the method of the present invention by adding a blood serum sample to a tungstic acidphosphotungstic acid mixture in the first compartment, attaching this compartment by filter means to a second compartment of aqueous sodium carbonate, centrifuging the assembly to cause the contents of the first compartment to filter into the second compartment to form upon agitation, a colored fluid, and measuring the optical density of the colored fluid, which optical density may be correlated with the concentration of uric acid.
To eliminate pouring of the colored fluid into a suitable cuvctte for optical density measurement, it is preferred that the container in which the colored fluid is formed by transparent and adapted for use in standard spectrophotometers, as in the apparatus hereinafter described.
The filter means of the present invention may be of any material which is resistant to the reagents employed in the desired determination; e.g. the filter means must not undergo significant chemical degradation nor significantly adsorb chemicals which pass therethrough during centrifugation. When connected to One container, the filter means must be sufficiently compact to prevent the flow of reagents therethrough under normal gravitational force (e.g., when assemblying the apparatus), and must be sufficiently porous to permit the passage of such reagents therethrough under a force substantially greater than the normal gravitational force (e.g. during centrifugation). It is believed that when the filter means is connected to one of the containers and the assembly is inverted to bring the fluid contents of the container into contact with the filter, a partial vacuum is created in the container as a small amount of the fluid contained therein seeps into the filter. The pressure across the filter is thereby equalized, preventing transfer of the fluid therethrough. During centrifugation, a large pressure differential develops across the filter means which causes substantially complete transfer therethrough of the fluid into the container furthest from the axis of rotation of the centrifuge.
Filters made from chemically-resistant polymers (e.g. polyolefins, fluorocarbon polymers, etc.) are useful in the method and apparatus of the present invention. Filters prepared from polyolefin fibers, especially polypropylene fibers, are preferred. It is desirable that such fibers have high surface/volume ratios. Fibers produced by the method described in U.S. Naval Research Laboratory Report No. 111,437, dated Apr. 15, 1954 and entitled Manufacture of Superfine Organic Fibers have been found to be especially useful and are preferred for filters of the present invention. Fibers produced by this method hereinafter are referred to as superfine fibers.
The filters of the present invention are preferably capable of removing precipitated protein from fluids passing therethrough during centrifugation. Protein is precipitated from blood serum, for example, when blood serum is mixed into the ferric chloride-acetic acid reagent used in total" cholesterol determinations. Haziness caused by precipitated protein in the colored fluids obtained by the above-mentioned total cholesterol methods can noticeably but not seriously disturb accuracy. Polypropylene fiber filters may be provided which filter out precipitated protein, however, by treating the filters with tap water having an analysis such as that referred to in Example 1 below. By use of such treated filters in the total cholesterol method of the present invention, excellent accuracy and precision may be obtained.
An apparatus for practicing the method of the present invention is shown in FIGS. 1-3 as assembled immediately prior to centrifugation.
Referring now to FIG. 1, a pair of glass vials 10 and 12 are provided containing respectively, a premeasured solution 14 capable of extracting a blood chemical from a blood derivative to form therewith a component capable of reacting with a test material to form a colored fluid, and a pre-measured test material 16 capable of forming with said component a colored fluid having an optical density dependent upon the concentration of the blood chemical in the blood derivative. The open ends 18 and 20 of the vials 10 and 12 are provided with threaded outer diameters 22 and 24 respectively. Prior to use, the glass vials 10 and 12 containing the solution 14 and the test material 16 are provided with threaded vial caps (not shown) for storage. Prior to centrifugation, as shown in FIG. 1, the vials 10 and 12 are connected by a connector member 26, the threaded ends 22 and 24 of the vials 10 and 12 threadingly engaging the threaded inner diameters 28 and 30 respectively of the open ends 32 and 34 of the connector member 26 to provide liquid-tight seals therewith. The connector member 26 is provided with an inner surface 35 having at a point 36 along its length a support means 38, such as a perforated transverse diaphragm,
against which is snugly seated a filter 40 which is chemically resistant to said solution 14 and to said test material 16. Upon centrifugation, the filter 40 is restrained from travel through the connector member 26 in the direction indicated by the arrow 42 by means of the perforated transverse diaphragm 38, which diaphragm readily permits, however, the passage of fluid material. The filter 40 is air permeable and, when joined to one or both of the vials 10 and 12, is capable of preventing the passage therethrough of either the component formed in vial 10 or the test material 16 under the normal force of gravity and is further capable of permitting passage of the component or the material 16 therethrough under a force substantially greater than the normal force of gravity.
Using the above-described apparatus, the method of the present invention may be practiced as follows, the blood chemical to be determined being total cholesteroli The protective vial cap is removed from the vial 10 which contains 3.0 ml. of a 0.5% solution of ferric chloride in glacial acetic acid, and 0.020 ml. of blood serum is deposited therein. The end 32 of connector member 26, having therein a filter 40, is threaded snugly onto the open end of the vial 10.
The assembly is inverted and is threaded onto the open end of a vial 12 containing 2.0 ml. of concentrated sulfuric acid. The total assembly is then placed in a centrifuge, the vial 10 being nearest the center of rotation thereof, and is centrifuged to cause the contents of the vial 10 to filter into the sulfuric acid. The two layers which are formed in the vial 12 are mixed by gentle agitation to form a colored fluid. After standing for 10 minutes, the vial 12 (still connected to the vial 10 by the connector member 26) is placed in a standard spectrophotometer and the optical density (O.D.) is measured and is correlated with the concentration of cholesterol by comparison with a graph of D. versus cholesterol concentration, which graph had been prepared by performing this procedure with standard solutions containing known concentrations of cholesterol.
The following examples are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention.
EXAMPLE 1 Three acetic acid test solutions containing 100, 200 and 300 mg. of cholesterol per 100 ml. of solution, respectively, were prepared by dissolving known quantities of solid cholesterol (Matheson, Coleman and Bell Co.) in acetic acid. Each solution was tested in the illustrated apparatus as follows:
0.020 ml. of test solution was added to 3.0 ml. of glacial acetic acid containing 0.05% (w./v.) of FeCl '6H O in a first threaded glass vial. Filter means identical to that illustrated in FIG. 1 was snugly threaded unto the glass vial, which filter means employed a filter pad which was prepared as follows:
Superfine polypropylene fibers, 95% of which were greater than 2.9 in diameter and 5% of which were greater than 17.5,u. in diameter, were formed into a mat which was treated successively with methyl ethyl ketone and with tap water of pH 8.2-8.7, the tap water (city water supply, St. Paul, Minn.) having an analysis essentially the same as that reported in Durfor and Becker, Public Water Supplies of the 100 Largest Cities in the United States, 1962, US. Government Printing Oflice, 1964, p. 222. Filter pads of the desired diameter were then cut from the treated mat.
The glass vial-filter means assembly was then inverted and threaded onto a second transparent glass vial of 13.25 mm. average inner diameter which contained 2.0 ml. of concentrated sulfurc acid. The apparatus was then centrifuged at a speed of 2000 revolutions per minute for 3 minutes in an International Centrifuge Model CL (International Equipment Co.) centrifuge. During centrifugation, the filter pad assumed a position 2.75 inches distant from the axis of rotation of the centrifuge. The force on the filter pad was calculated to be 170 times the normal force of gravity. The filtrate which passed through the filter was transparent and formed a separate layer upon the sulfuric acid. The layers were mixed by gentle agitation of the apparatus, during which time the temperature of the resulting solution rose to about 60 C. and a red color developed therein.
After standing for 10 minutes at room temperature, the second glass vial (still attached to the first glass vial by the filter means) was placed in the cuvette well of a Coleman Junion Colorimeter (Coleman Instruments Co.) using a simple adaptor, and the optical density of the colored solution was measured at 560 m as follows:
cholesterol/ ml.: Optical density By plotting concentration versus optical density on rectangular coordinate paper, a standard curve Was obtained which was essentially linear.
The above procedure was repeated using samples of blood serum containing unknown quantities of cholesterol. The optical density of each colored solution was correlated with the cholesterol concentration by use of the standard curve obtained above. Concurrent tests were performed on these blood serum samples by the widelyused method of Abell et al., as reported by Henry, R. J., Clinical Chemistry: Principles and Techniques, Harper and Row, New York, 1965, pp. 852855. Results are reported in Table I.
TABLE I Mg. cholesterol/100 m1. of blood serum The data of Abell et al., were plotted on rectangular coordinate paper against the corresponding data resulting from the method of the present invention. A line was drawn through the data points by the method of least squares and was found to have a slope of 1.023. A correlation coefficient (Dixon, W. J. and Massey, F. 1., Introduction to Statistical Analysis, 2d ed. McGraw-Hill, New York, 1957, Chap. 11) of 0.286 mg./ 100 ml. was
computed from the above data, indicating excellent corre- I lation between the method of the present invention and the method of Abell et al.
EXAMPLES 25 By using the general procedure of Example 1, quantitative determinations of uric acid, protein, urea, and glucose may be obtained. Test parameters for these determinations are provided in Table II.
TABLE II.-REACTANTS Ex. Blood chemical 1st vial 2d vial at denslty measured Reference 2 Uric acid (0.1 ml. of blood Tungstic acid/phospho- 14% aqueous N fizCOa 660 m (after standin serum). It1uln)gstate acid 1 3.05 0.75 ml.). 15 minutes). g fi l'n rlf %8gllgl 2 l 2l 3 Protein (0.070 ml. of 3.0% aqueous NaOH Bluret reagent (0.7 545 m afte blood serum). solution (3.5 ml.). m1.) (see reference). 15 marines) standing 8 3,, gi fg gibg 4 Urea (0.5 ml. of blood 10% aqueous tnchloro- Modified Ehrlich reagent 1 425 my, im standing 4 Levine J M et l Cli serum). acetic acid (3.0 ml.). (1.0 ml.). i t Chm;1 6i Glucose (0.050 ml. of 3% aqueous triehloro- 6% o-toluidme in glacial 625m (after boiling for Hultman 15 Nature 183' blood serum). acetic acid (1.0 ml.).
l Prepared by combining 0.75 ml. of an equal volume mixture 01067 N, H2804 and 10% aqueous sodium tn 1* I l R 1., Clinical Chemistry: Principles and Techniques, Harper and Row New Y k 1965 2 Prepared by combining 5.0 g. p-dimethylaminobenzaldehyde, 20 m1. concentration H01 and 80 ml. distilled water. p
stic acid reagent reported in Henry,
What is claimed is:
1. A method for the quantitative determination of a blood chemical comprising the sequential steps of (1) providing a first container of solution capable of extracting a blood chemical from a blood derivative to form a component capable of reacting with a test material to form a colored fluid having an optical density dependent upon the concentration of said blood chemical in said blood derivative;
(2) adding to said first container a sample of a blood derivative containing said blood chemical to form said component;
(3) providing a second container containing said test material;
(4) joining said containers with air-permeable filter means which is chemically resistant to and which normally separates the solution of said first container and the material of said second container, said filter means being capable of preventing the passage therethrough of one of said component and said material under normal gravitational force and being capable of permitting passage therethrough of said one of said component and said material under a force substantially greater than the normal force of gravity;
(5) centrifuging said joined containers to combine the contents thereof and to form, with agitation, a colored fluid; and
(6) measuring the optical density of said colored fluid.
2. The method of claim 1, wherein said other container is substantially transparent to light and wherein said optical density of said colored fluid is measured within said transparent other container.
3. The method of claim 2, wherein said blood chemical is cholesterol, wherein said solution consists of acetic acid and ferric chloride, and wherein said test material is concentrated sulfuric acid.
4. A method for measuring the concentration of cholesterol in a blood derivative comprising (a) providing a first container of premeasured solution of ferric chloride and acetic acid;
(b) providing a transparent second container of a premeasured amount of concentrated sulfuric acid;
acetic acid (3.0 ml.).
10 minutes in 2d vial and cooling). 108 (1959).
ml. of the phosphotung- (0) adding to said first container a sample of a blood derivative containing cholesterol to form a component reactive with concentrated sulfuric acid to form a colored fluid;
(d) joining said containers with air-permeable filter rneans chemically resistant to and normally separatrng said solution and said sulfuric acid, said filter means being capable of preventing the passage of said component therethrough under normal gravitational force and being further capable of permitting passage therethrough of said component under a force substantially greater than the normal force of gravity;
(e) centrifuging said joined containers in a centrifuge with said first container being nearer the axis of rotation of said centrifuge than said second container to cause filtration of said component through said filter to form separate layers in said second container;
(f) rnxing said layers to provide a colored solution;
(g) measuring within said second container the optical density of said colored solution.
References Cited UNITED STATES PATENTS 93 8,279 10/ 1909 Schultze 23292 X 2,110,237 3/1938 Parsons 23292 X 3,215,500 11/1965 Bittner 23259 3,449,081 6/1969 Hughes 23253 2,129,516 9/1938 Wood 23231 OTHER REFERENCES Welcher, F. 1.: Standard Methods of Chemical Analys1s,, vol. II, Part A, pp. 1088-9 (1963).
JOSEPH SCOVRONEK, Primary Examiner R. M. REESE, Assistant Examiner US. Cl. X.R.