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
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/721—Haemoglobin
  • G01N33/725—Haemoglobin using peroxidative activity

Definitions

• this invention is concerned with a diagnostic test which is useful for the qualitative detection and quantitative determination of blood in body fluids and products of elimination such as urine, vomitus, gastro-intestinal contents, cerebrospinal fluids and feces.
• this invention is concerned with a highly sensitive reagent composition for the detection of occult blood which may be incorporated upon a bibulous carrier..
• Blood is found in the gastric contents and in vomitus in conditions associated with erosion of gastric and intestinal mucuous membranes and, for example, ulcerous and carcinogenic conditions.
• the regular and frequent occurrence of occult blood is suggestive of, for example, gastro-intestinal cancer, gastric or duodenal ulcers or hemorrhoids. Quite often, hemorrhage is so slight that it is no possible to detect the presence of blood by microscopic examination. Hence, a sensitive and specific diagnostic test for occult blood. is highly desirable.
• the presence of blood cells or blood pigments may be indicative of a number of abnormal conditions, for example, typhus, scurvy, purpura, pyemia, nephritis, third degree burns, carcinogenic conditions of the urinary system, and as a result of the action of various hemolytic toxins and the like.
• a simple and economical diagnostic test composition is provided which may advantageously be used with ease by hospital and clinical personnel untrained in laboratory technique as well .as the skilled laboratory technician and physician.
• the inventive concept of the instant application is based on the catalytic activity of the prosthetic groups present in blood.
• the catalytically active substances identified. in hemoglobin belong to the general class of hemoproteins, conjugate proteins, all of which have the same prosthetic groups, iron protoporphyrin or heme.
• This prosthetic group has the ability to catalyze the oxidation of certain compounds by peroxides such as hydrogen peroxide, metal peroxides and organic peroxides. If the compound is an indicator or dye pre 3,290,117 Patented Dec. 6, 1966 cursor, colorless until it becomes oxidized and colored in its oxidized form, then the presence of the catalytic activity and hence the presence of blood is indicated by color formation.
• the rapidity of the color change and the depth or density of the color when compared to a set of standards is a means of the quantitative estimation of the blood present.
• quinoline derivatives found to be satisiactory as potentiating agents in the formulation of this where R may be hydrogen, methyl or hydroxy; R may be hydrogen, methyl or thiophenyl; R may be hydrogen or methyl or the substituted quinuclidine radical,
• suitable potentiat-ing agents include quinoline; quinine; cinchonine; 6-methoxyquinoline; 4,6-dimethylquinoline; 6-methylquinoline; 7-methylquinoline; 2,6-dimethylquinoline; Z-methylquinoline (quinaldine); 8- amino-6-methoxyquinoline; 6-methoxy-3-phenylthioquino-' line; 8-methylquinoline; 2,3-dimethylquinoline 2-quinolinol; 2-methyl-8-quinolinol and 8-quino'1-r'nol.
• each of the occult blood tests aforementioned may be improved by the addition of the quinoline or substituted quinoline potentiating agent.
• the potentiating agent may be mixed with the dry compositions or tablets of U.S. Patent 2,290,436 to produce a diagnostic composition having greater sensitivity than the mixture of indicator peroxide and acidic material disclosed in the patent.
• the tablets disclosed in U.S. Patent 2,799,660 comprising indicator peroxide buffer and effervescent couple and color contrast agent may be similarly improved by use of the potentiating agents of this invention.
• the blood test of U.S. Patent 2,838,377 comprising an indicator, a peroxide and a bufier incorporated in a closed envelope formed of an electrolyte permeable sheet material may be similarly improved by the utilization of one or more of the various potentiating agents described above.
• the occult blood tests described in U.S. Patent 3,012,- 976 comprise an organic hydroperoxide, an indicator and a buffer and may be improved by the inclusion therein of the potentiating agent of the instant invention.
• the encapsulated compositions of U.S. Patent 3,092,463 may be likewise improved by use of a potentiating agent of the quinoline type.
• the organic hydroperoxides of U.S. 3,092,464 are similarly improved.
• a group of organic hydroperoxides having pronounced sensitivity in an indicator reaction may be encapsulated or entrapped with a colloidal material such as gelatin and the gelatin hardened by fixing with a dialdehyde polysaccharide.
• a colloidal material such as gelatin
• a dialdehyde polysaccharide a colloidal material
• compositions of this invention may be prepared in solution or tablet form or may be used to impregnate a bibulous material such as paper, wood, fiber or the like, having any desired size or shape. Such products will undergo a distinct color change when contacted with an occult blood-containing specimen.
• the group of organic hydroperoxides mentioned above which have been found to have the pronounced sensitivity which is required for the indicator reaction to be utilized in the diagnostic compositions of this invention comprise a group of materials including cu-mene hydroperoxide; diisopropylbenzene hydroperoxide; paramethane hydroperoxide; and 2,5-dimethy-lhexane, 2,5-dihydroperoxide; to mention a few of the significant members of this group.
• Other organic hydroperoxides of similar structure may likewise be used if desired.
• Organic hydroperoxides while producing outstanding results in various occult blood diagnostic compositions, have been found to be relatively unstable when mixed with the various other materials which are required in the provision of diagnostic compositions of this nature.
• a novel encapsulating material is provide-d for the organic hydroperoxide.
• This encapsulating material may be comprised of various protein or polysaccharide materials such as gelatin, algin, carrageenin, casein, albumin or other materials of this nature. These proteinaceous or 'polysaccharous materials are applied to the test compositions and then hardened by means of a fixing process which involves treating with a dialdehyde polysa-ccharide which is effective for this purpose.
• buffering systems useful in the composition of this invention are tartrate, phosphate, phthalate, citrate and acetate buffer.
• the preferred range of hydrogen ion concentration to which the composition is buifered is about pH 4 to pH 7.
• dialdehyde polysaccharides sometimes known as periodate oxidized polysaccharides, for example, dialdehyde starch
• Dialdehyde polysaccharides may be prepared by the well-known oxidation of polysaccharides with periodic acid. This preparation is illustrated by the conversion of starch to dialdehyde starch using periodic acid as the oxidizing agent in accordance with the below set-out equation wherein x stands for the number of repeating units in a molecule which may range from 20 to several thousand.
• OHzOH CHzOH hyde polysaccharides which are from about 50% to oxidized, that is, in the case of dialdehyde starch, those wherein 50 to 100 per 100 of the original anhydroglucose units have been converted to dialdehyde units such as by periodate oxidation.
• the dialdehyde polysaccharide is used in the form of an aqueous dispersion which may be readily prepared by adding the dialdehyde polysaccharide in the desired concentration, usually from about 0.25% to about 1.5%, to tap water or to a buffer solution and then mixing the aqueous dispersion with warming until a relatively homogenous dispersion is obtained.
• the dialdehyde polysaccharide may be used in whatever concentration is required to satisfactorily harden the encapsulating material to provide the desired stability features. The precise concentration to be used will be pointed out more particularly hereafter.
• emulsifying agent for example, acacia mucilage
• Other emulsifying agents which may be used include polyvinyl alcohol, gum arabic, carboxy vinyl polymer, and the like.
• a surfactant, or wetting agent may also be used.
• sodium lauryl sulfate is used as a wetting agent, however, dioctyl sodium sulfosuccinate for example is also satisfactory.
• An emulsifying agent when used serves to lower the surface tension of the oily organic hydroperoxide and forms a film around each individual oil droplet.
• the surfactant assists in this regard and, in addition, produces an even diffusion of color on the diagnostic stick.
• the resulting primary emulsion is buffered by means of an'appropriate buffer as discussed hereinabove.
• the buffer in dilute form is first mixed with the material which is to form the capsule for the organic peroxide, for example, gelatin, before addition to the hydroperoxide emulsion. If the concentrated buffer is used with the encapsulating agent there is a tendency to precipitate the encapsulating agent prior to completion of the encapsulation. After the organic hydroperoxide has been entrapped or encapsulated, the concentrated buifer is added which has the effect of setting the encapsulation while controlling the pH and preventing false positives in testing.
• aqueous dispersion of the dialdehyde polysaccharide is next added to serve as a fixing agent for the encapsulating material.
• the encapsulation material which is present in the film around the oily organic hydroperoxide droplets is thereby fixed by the dialdehyde polysaccharide, resulting in a more stable preparation.
• orthotolidine is the preferred indicator for the diagnostic compositions of this invention
• various other indicator materials may be used so long as they satisfy the requirements pointed out above, namely, that they undergo a color change in the presence of an oxygen source and the blood for which the unknown material is being tested.
• Such indicators comprise a variety of organic materials, principally those of aniline and phenol derivation.
• quinoline itself or quinolines substituted in the 4-, 6- or 7-positions are used. Because of availability, economic reasons, and suitable physical properties, quinine has been found quite useful.
• organic solvents found useful in the composition and methods of this invention are chloroform, ethylene dichloride, benzene and ethyl acetate.
• the novel compositions of this invention may' be prepared in tablet form, a convenient tablet size being about /8" thick and about A" in diameter.
• a drop of the material to be tested in the case of fecesan aqueous suspension of the specimen
• a reagent tablet is placed in the center of the drop and activated by the adition of two drops of water to the tablet.
• a positive test With a positive test, a ring of color appears on the filter paper surrounding the tablet, the color intensity varying, dependent upon the concentration of blood in the specimen. The color developed is then compared with a calibrated color chart to determine the concentration.
• the dry composition may be added to a specific amount of water and mixed with the material to be tested. In the presence of occult blood, a color will be obtained. An estimation of the amount of occult blood present in the specimen may be made by comparing the color developed with calibrated standards.
• compositions of the diagnostic material provided by this invention may vary within fairly wide limits in accordance with the general teachings set forth above.
• Buffer II (489.0 g. sodium citrate plus 111.0 g. citric acid plus 1% liters water) was then added with mixing.
• EXAMPLES 3 and 4 The testing reagents were prepared in accordance with Example 1, the quinine alkaloid ingredient being increased to 20 mg. and 50 mg. per 20 ml. respectively.
• EXAMPLE 7 The hydroperoxide solution was prepared in accordance with that of Example 1.
• the indicator solution was prepared to contain mg. o-tolidine, 10 mg. quinine alkaloid and 2 mg. polyoxyethylenenonylphenol per 20 ml. chloroform.
• EXAMPLE 8 A hydroperoxide solution was prepared in accordance with that of Example 1.
• the indicator solution was prepared to contain 80 mg. o-tolidine, 5 mg. 6-methylquinoline and 2 mg. polyoxyethylenenonylphenol per 20 ml.
• EXAMPLE 9 A hydroperoxide solution was prepared in accordance wtih that of Example 1.
• the indicator solution was prepared to contain 80 mg. o-tolidine, 5 mg. 6-methylquinoline and 2 mg. polyoxyethylenenonylphenol per 20 mg.
• EXAMPLE 10 A hydroperoxide solution was prepared in accordance with that of Example 1.
• the indicator solution was prepared to contain 80 mg. o-tolidine, 5 mg. 7-methylquinoline and 2 mg. polyoxyethylenenonylphenol per 20 ml.
• EXAMPLE 1 1 A hydroperoxide solution was prepared in accordance with that of Example 1.
• the indicator solution was prepared to contain 80 mg. o-tolidine, 5 mg. 4,6-dimethylquinoline and 2 mg. polyoxyethylenenonylphenol per 20 ml.
• EXAMPLE 12 A hydroperoxide solution was prepared in accordance with that of Example 1.
• the indicator solution was prepared to contain 80 mg. o-tolidine, 5 mg. 2,6-dimethylquinoline and 2 mg. polyoxyethylenenonylphenol.
• EXAMPLE 13 amount of o-tolidine in the formulation for the reason that o-tolidine is less soluble in benzene than in chloroform or ethylene dichloride. While it is not essential, it was unexpectedly discovered by observation under ultraviolet light that the addition of a surface-active agent, for example, polyoxyethylenenonylphenol, to the indicator solution resulted in a more even application of the color indicator and quinine alkaloid when prepared in a strip or stick form. It was found that the amount of surfaceactive agent could be varied over a range between 1 and 20 mg. per 20 ml., the preferred amount being in the lower end of the range.
• a surface-active agent for example, polyoxyethylenenonylphenol
• Bibulous sticks that is, absorbent paper out into narrow strips having dimensions of about 3" x /5" x 0.029, were dipped into the hydroperoxide solution followed by drying in a drying tunnel at a temperature of about 100 C.
• the dried strips were subsequently heated in an oven at 70 C. for 8 to 16 hours to remove excess cumene hydroperoxide.
• the strips were similarly dipped into the indicator solution and dried in a 70 C. oven or a 50 C. drying tunnel.
• the finished impregnated strips were white to cream in color.
• Table 1 sets out the results of 114 urine examinations from hospitalized patients, comparing the improved diagnostic test of this invention with prior known formulations.
• TAB LE 1 Number Composition Applicants of Urines Cells per High Without Po Improved in Classi- Power Field tentiating Testing Comfication Agent position 62 Occasional 1 55 negatives 48 negatives 4 traces 5 traces 3 small 7 small 2 moderate 26 1-5 19 negatives 10 negatives 2 traces 1 trace 4 small 8 small 1 moderate 6 moderate 1 large 11 5-10 5 negatives 1 negative trace trace 2 small 3 small 4 moderate 4 moderate large 7 1020 3 negatives negative 1 trace 0 trace 2 small 3 small 1 moderate 4 moderate 8 20-50 2 negatives 0 negative 2 traces trace 2 small 3 small 2 moderate 3 moderate 2 large EXAMPLE 14 Benzidine method To 2 ml. of urine add 3 ml. saturated benzidine in glacial acetic acid (4 .g.O ml.) add 1 ml. of 3% hydrogen peroxide mix. A green to blue color indicates a positive reaction.
• R is a member selected from the group consisting of hydrogen, methyl and methoxy
• R is a member selected from the group consisting of hydrogen and methyl
• R is a member selected from the group consisting of hydrogen, hy'droxy, methyl and amino.
• composition of claim 1 wherein the potentiating agent is quinine.
• composition of claim 1 wherein the potentiating agent is 6-methoxyquinoline.
• composition of claim 1 wherein the potentiating agent is 6-methyl quinoline.
• composition of claim 1 wherein the potentiating agent is 7-methyl quinoline.
• composition of claim 1 wherein the potentiating agent is 4,6-dimethyl quinoline.
• composition of claim 1 ing agent is quinoline.
• composition of claim 1 wherein the organic hydroperoxide is a member selected from the group consisting of cumene hydroperoxide, diisopropylbenzene hydroperoxide, paramenthane hydroperoxide and 2,5-dimethylhexane-2,5-dihydroperoxide.
• a composition for detecting blood which comprises an indicator capable of being oxidized in the presence of the prosthetic group of hemoglobin with an accompanying color change, an oxidizing agent effective to oxidize said indicator and a potentiating agent of the formula wherein the potentiatthiophenyl; R is a member selected from the group consisting of hydrogen, methyl and the radical E R is a member selected from the group consisting of hydrogen, methyl and methoxy; R is a member selected from the group consisting of hydrogen and methyl; and R is a member selected from the group consisting of hydrogen, hydroxy, methyl and amino.
• composition according to claim 9 wherein the potentiating agent is a member selected from the group consisting of quinine, 6-methoxyquinoline, 6-methyl quinoline, 7-methyl quinoline, 4,6-dimethyl quinoline and quinoline.
• a test indicator for detecting occult blood in body fluids and excreta comprising a bibulous material containing therein the dried residue resulting from the deposition on said material of a mixture comprising a first aqueous solution of an organic hydroperoxide, a buffer capable of maintaining the pH of the material being tested within the range of about 4 to 7, gelatin and as a fixative for said gelatin a dialdehyde polysaccharide, and a second solution of an indicator capable of being oxidized by the hydroperoxide in the presence of the prosthetic group of hemoglobin with an accompanying color change, a potentiating agent of the formula 19
• R is a member selected from the group consisting of hydrogen, methyl and methoxy
• R is a member selected from the group consisting of hydrogen and methyl
• R is a member selected from the group consisting of hydrogen, hydroxy, methyl and amino and an organic solvent.
• test indicator of claim 11 wherein the potentiating agent is 6-methoxyquinoline.
• test indicator of claim 11 wherein the potentiating agent is 6-methyl quinoline.
• test indicator of claim 11 wherein the potentiating agent is 7-methyl quinoline.
• test indicator of claim 11 wherein the potentiating agent is 4,6-dimethyl quinoline.
• test indicator of claim 11 wherein the potentiating agent is quinoline.
• organic hydroperoxide is a member selected from the group consisting of cumene hydroperoxide, diisopropylbenzene hydroperoxide, paramethane hydroperoxide and 2,5-dirnethylheXane-2,5-dihydroperoxide.

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Citations (2)

• 0
  • NL NL126365D patent/NL126365C/xx active
• 1963
  • 1963-06-24 US US290253A patent/US3290117A/en not_active Expired - Lifetime
• 1964
  • 1964-06-11 DE DEM61333A patent/DE1242905B/de active Pending
  • 1964-06-23 BR BR160307/64A patent/BR6460307D0/pt unknown
  • 1964-06-23 SE SE7629/64A patent/SE300717B/xx unknown
  • 1964-06-23 CH CH817164A patent/CH446767A/de unknown
  • 1964-06-24 GB GB26150/64A patent/GB1057056A/en not_active Expired
  • 1964-06-24 BE BE649682A patent/BE649682A/xx unknown
  • 1964-06-24 DK DK317064AA patent/DK115507B/da unknown
  • 1964-06-24 NL NL6407185A patent/NL6407185A/xx unknown

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