WO2011123776A2 - Procédé de détection du sang occulte - Google Patents

Procédé de détection du sang occulte Download PDF

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WO2011123776A2
WO2011123776A2 PCT/US2011/030934 US2011030934W WO2011123776A2 WO 2011123776 A2 WO2011123776 A2 WO 2011123776A2 US 2011030934 W US2011030934 W US 2011030934W WO 2011123776 A2 WO2011123776 A2 WO 2011123776A2
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luminol
reagent
sodium
specimen
chemiluminescent
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PCT/US2011/030934
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WO2011123776A3 (fr
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B. Eugene Guthery
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Guthery B Eugene
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    • 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 relates to the field of the detection of occult blood in specimens, particularly in feces.
  • Colorectal cancer is common and may be fatal. It is the second-leading cause of death from cancer in the USA. In the absence of effective methods for the prevention of cancer, public health measures have been directed toward early detection.
  • the current methods of colorectal cancer screening in the general population include searching for occult blood in the stool, total colonoscopy, flexible sigmoidoscopy, and tests based on abnormal DNA found in fecal specimens.
  • Occult bleeding from the gastrointestinal tract may be detected by examination of the excreta for evidence of blood through the use of chemical laboratory techniques.
  • the presence of occult blood in the feces is important because it may indicate otherwise asymptomatic
  • gastrointestinal neoplasia and may also be helpful in the evaluation of gastrointestinal symptoms in the absence of visible bleeding. Colonoscopy and perhaps examination of the upper gastrointestinal tract with esophagogastroduodenoscopy (EGD) is strongly recommended for anyone with a positive fecal occult blood test (FBOT) on any fecal
  • Compliance in non-selected populations has been estimated to be less than 50%, at least partly because the techniques require patients themselves to first select and then smear their stool specimen onto a slide or a test-strip, a task most people find not only loathsome, but also technically difficult.
  • Such collection generally involves use of a device that will contact feces deposited in a toilet bowl in the usual manner.
  • a device is used to contact the feces, obtain a portion thereof, and is used to streak a sample of the feces on a test strip which will be sent to a laboratory or physician's office for analysis.
  • This process is also an unwieldy task, especially if the feces have sunk to the bottom of the toilet bowl.
  • Sampling stool from the toilet bowl also introduces error because blood leaches from the fecal surface into the surrounding water which may not be collected for testing, possibly causing a false negative.
  • Another testing problem lies with the nature of fecal specimens.
  • Fecal specimens are not homogeneous and some portions may have occult blood while others do not.
  • a standard FOBT that requires sampling of a small portion is useful, providing the small specimen of stool tested actually contains a representative amount of the blood shed in the gastrointestinal tract by the patient. This implies that a single standard fecal occult blood test that requires sampling may possibly give a false negative result simply because the wrong portion of the fecal specimen was tested. Multiple tests may be required to ensure that any occult bleeding is discovered.
  • the intermittent nature of gastrointestinal bleeding confounds the problem since the patient must choose the correct portion of stool to yield an accurate result.
  • Luminol preparations have been utilized for the detection of blood at crime scenes or for tracking wounded animals.
  • Japanese patent 05256748 discloses use of an ultrasonic vibrator to disrupt the fecal specimen in the toilet bowl. Next, a luminol preparation and peroxides are to be added to the disrupted specimen. Any luminescence which results is then determined with a photodiode sensing section on the vibrator and read with a hand-held meter.
  • Japanese patent 05002017 discloses the use of a luminol preparation and hydrogen peroxide. This preparation is to be poured into a commode and any subsequent luminescence is determined with a photodiode or photomultiplier.
  • Japanese patent 05273206 A discloses a similar process to Japanese patent 05002017 except that EDTA is to be added prior to adding luminol in an attempt to sequester any trace metals in the water containing the feces to try to prevent false positives due to trace metals like iron or copper in the water supply.
  • FIG. 1 shows a flow chart for a method of use of the fecal occult blood detection system of the present invention.
  • FIG. 2 shows the proposed reaction pathway for the luminol reagent.
  • FIG. 3 shows the results of time vs. chemiluminescence for luminol
  • the method employs a reagent comprising an agent selected from the group consisting of purified sodium luminol, purified potassium luminol, cyclic hydrazides and acyclic hydrazides, preferably the sodium or potassium salts of luminol (5-amino-2,3-dihydro 1 ,4-phthalazinedione; o-amino-phthalyhydrazide) and/or the sodium or potassium salts of benzo (ghi)perylene-1 ,2-dicarboxylic acid.
  • an agent selected from the group consisting of purified sodium luminol, purified potassium luminol, cyclic hydrazides and acyclic hydrazides, preferably the sodium or potassium salts of luminol (5-amino-2,3-dihydro 1 ,4-phthalazinedione; o-amino-phthalyhydrazide) and/or the sodium or potassium salts of benzo (ghi)perylene
  • the method of the invention provides the following advantages as a screening test for fecal occult blood: 1 ) it is simple to use, 2) it is cost effective, 3) it minimizes the percentage of false-negative results (sensitivity), and 4) it has a low incidence of false- positive results (specificity).
  • the sensitivity of a fecal occult blood test determines its benefit to the user while the specificity of the test determines its cost effectiveness in terms of the need to screen patients who have positive tests. Since this method requires no fecal specimen sampling, compliance may be increased, thereby overcoming an obstacle found with previous methods.
  • the sensitivity and specificity of the invention exceeds that of currently reported "throw-in-the-bowl" tests, thereby meeting another need in the fecal occult blood testing arena.
  • the sensitivity exceeds that of the immunochemical methods as well.
  • the method of the invention also does not require the use of any device, such as a photodiode or photomultiplier to read the results, thus meeting yet another need for fecal occult blood testing.
  • Figure 1 shows a schematic for the method of the current invention for the detection of occult blood.
  • a specimen is deposited, usually through direct defecation, into a suitable receptacle, such as a commode bowl, a bedpan or other receptacle.
  • a suitable receptacle such as a commode bowl, a bedpan or other receptacle.
  • Test reagents are added sequentially to the receptacle containing the specimen, the lights extinguished, and the presence or absence of chemiluminescence is noted. Presence of any chemiluminescence in the receptacle with the specimen is indicative of occult blood.
  • the schematic for Figure 1 relates to the use of the method for the present invention with a toilet (commode) bowl as the preferred receptacle for the specimen, but it is understood that other receptacles can be used as stated above.
  • a negative control test should be performed in the following manner.
  • a sufficient quantity of an oxidizing solution as taught herein is poured into the water of a commode (toilet) bowl. Said quantity of oxidizing solution is of an adequate amount for the approximately 1800 ml_ of water contained in a typical toilet bowl.
  • a predetermined quantity of a chemiluminescent reagent, as taught herein is poured into the commode.
  • the room light is turned out, and the toilet bowl is observed.
  • any neon-like glow observed from the commode water with this procedure indicates residual toilet bowl cleaners or trace metals from the plumbing in the commode water. If a blue glowing light occurs with the use of only the oxidizing solution and the chemiluminescent solution (1 E), the patient is instructed to stop the test, and then is further instructed in 1 F to avoid using toilet bowl cleaners, to remove any cleaning products that may be present in the commode tank, and to flush the commode twice a day over a three day period, after which time, the negative control test should be repeated. If chemiluminescence is again present, the cause is the presence of excess trace metals in the water supply and the test for occult blood should not be performed in this commode.
  • the patient must defecate in a bedpan or other receptacle to test for fecal occult blood.
  • Approximately one liter of deionized water (enough to cover the fecal specimen) should be poured over the fecal specimen in the receptacle.
  • the reagents are added per Step 3 of FIG. 1 as if the test were being performed in a toilet bowl. If an alternate receptacle is utilized, the negative and positive control steps should be omitted.
  • the control is considered to be acceptably negative (1 D), and the patient is instructed to flush the toilet twice to remove the reagents from the toilet bowl before proceeding to Testing Procedure Step 2 - Positive Control.
  • Any neon-like blue glowing light that may occur when flushing the commode in the dark is to be ignored. Even though a negative control test can be completely negative, a brilliant, blue glowing light may be produced when the commode is flushed. This is due to trace metals from the toilet bowl tank mixing with the reagents and does not represent occult blood in the fecal specimen.
  • Step 2 Positive Control. If the negative control test is satisfactorily negative, a positive control test is preferably performed to illustrate to the user how a positive test should appear.
  • the positive control may be oxidizing compounds like hypochlorite, iodine, or iodophors.
  • Dried beef blood functions well as a positive control, as does purified human hemoglobin that has been tested free of HIV and hepatitis. Hemin or Sodium and potassium salts of Hemin (Frontier Scientific, Inc. in, Logan, Utah, USA) are the positive controls of choice.
  • an adequate quantity of an oxidizing solution (2A) is added to the commode water.
  • a positive control agent for example, one drop of a Hemin solution from Porphyrin Products, Inc. containing 150 mg hemin/mL dissolved in ethanol
  • the chemiluminescent or chemifluorescent compound of 2C is sprayed or poured into the toilet bowl water and the room is darkened (2D) prior to reading the test.
  • the chemiluminescence or chemifluorescence detected with the naked eye in the darkened room is noted by the user.
  • Step 3 Patient Sample.
  • the patient defecates or micturates (3A) into the toilet bowl.
  • 3B an adequate quantity of the oxidizing solution is added to the toilet bowl water.
  • an adequate volume of the oxidizing solution is added to the toilet bowl water.
  • chemiluminescent or chemifluorescent compound 3C
  • chemiluminescent or chemifluorescent compound 3C
  • the fecal or urine sample contains blood or its degradation products like heme, hemoglobin, protoporphyrin IX, or iron, there is an immediate dramatic production of a neon-like blue light visible to the naked eye in the darkened room, which may be seen without the use of photodiodes or photomultipliers.
  • the amount of light production depends on the amount of heme, hemoglobin,
  • protoporphyrin IX or iron in the toilet bowl water. If any amount of neon-like glowing blue light is seen, the test is positive (3F) for occult blood and the patient is instructed to contact a physician regarding follow-up testing, such as a colonoscopy. (3G). If the sample in the toilet bowl water does not contain heme, hemoglobin, protoporphyrin IX, or iron, there will be no light visible and the test is considered negative for occult blood (3E).
  • the preferred chemiluminescent and/or chemi-fluorescent compound is sodium luminol.
  • this invention requires the application of an oxidizing compound either just prior or subsequent to micturition or defecation, followed by application of a chemiluminescent or chemifluorescent compound.
  • the oxidizing compound and chemiluminescent or chemifluorescent components may be mixed together just prior to application to the urine and/or fecal specimen.
  • the reaction may also be achieved by simply pouring the chemicals into the device containing the specimen if there is enough water in the device..
  • One mode of applying the required chemicals is with a bottle fitted with a spray nozzle.
  • the volume delivered by the spray nozzle should be such that approximately five to ten actuations of the spray nozzle are needed to apply an adequate quantity of each chemical for each test.
  • a preferred method is to simply pour an adequate volume of hydrogen peroxide containing a chelator into the container holding the specimen followed by pouring an adequate volume of the chemiluminescent reagent.
  • the test for occult blood of the current invention may also be used on aspirates of gastric juice obtained via naso-gastric tubes.
  • the hydrogen peroxide and sodium luminol are poured into the receptacle containing the specimen, the light is turned off and any chemiluminescence is easily determined.
  • the test for occult blood of the current invention may also be used on sputum samples.
  • a patient would take an inhalation treatment with hypertonic saline (three percent sodium chloride) administered via a regular nebulizer or ultrasonic nebulizer.
  • the hypertonic saline would induce water into the bronchial tubes and induce sputum production.
  • the sputum is saved in a sterile cup.
  • the hydrogen peroxide and sodium Luminol are poured into the cup.
  • the light is turned off.
  • the production of chemiluminescence indicates occult blood in the sputum.
  • the patient should be referred for bronchoscopy. This may lead to an earlier detection of bronchial carcinoma.
  • the test for occult blood of the current invention may also be used on urine samples.
  • the urine could be tested in the commode water as one would test for occult blood on fecal samples.
  • a one to two ounce urine specimen could have the oxidizing agent and Sodium Luminol added to the sterile urine cup. The light is turned off. The production of chemiluminescent indicates occult blood in the urine.
  • the patient will require an intravenous pyelogram and perhaps cystoscopy.
  • the volume of the reagents necessary for the reaction optimization will vary depending on the volume of the receptacle holding the specimen.
  • the preferred range of volume of the oxidizing reagent for a standard commode bowl is 10-40 ml_ of the 5 % hydrogen peroxide - chelator solution, with 30 ml_ being most preferred.
  • the preferred range of volume of the chemiluminescent reagent for a standard commode bowl is 10- 50 ml_ of the reagent containing 2 mg/mL purified luminol in 75 mg/mL sodium
  • the fecal (stool) specimen contains peroxidases from plants, or catalases then there may be very minuscule light output from a multitude sites on the stool surface. Blood produces a dramatic neon-like blue light output which is sustained and easily seen in a dark room, whereas peroxidases from other sources produce only minuscule, if any, light output.
  • the light output from heme, hemoglobin, protoporphyrin IX, or iron is both dramatic and prolonged. The light output may last as long as ten minutes. The light output may also be reproduced by a second application of the chemiluminescent reagent disclosed herein.
  • the method of the invention overcomes a deficiency in other tests for fecal occult blood, i.e., guaiac, which are accompanied by an unacceptably large number of false positives due to dietary peroxidases which are common in onion, bell peppers and radishes, for example.
  • Human hemoglobin is a pseudo-peroxidase.
  • the false- positives caused by dietary peroxidases are because the ideal pH for performing guaiac tests and detecting dietary sources of peroxidases overlap.
  • an oxidizing reagent is added to the receptacle holding the specimen.
  • An oxidizing agent is used to lyse any red blood cells present in the specimen in order to release the hemoglobin.
  • the oxidizing compound serves as a catalyst for luminol.
  • the oxidizing reagent is selected from inorganic peroxides, organic peroxides, and mixtures thereof.
  • the oxidizing reagent must further comprise a chelator to bind metal ions which may be present in the toilet water either before or after deposition of the specimen.
  • the same oxidizing reagent is also used in the negative and positive control tests in steps 1A and 2A.
  • inorganic peroxides which may be used are hydrogen peroxide, sodium peroxycarbonate, and sodium perborate or mixtures thereof.
  • Preferred inorganic peroxides are hydrogen peroxide, sodium peroxycarbonate and sodium perborate.
  • the preferred concentration is between three and five percent. Three percent hydrogen peroxide is most preferred.
  • peroxides may influence the choice of chelator utilized as is explained further below.
  • Organic peroxides which may be used include diacyl peroxides, ketone peroxides, peroxydicarbonates, peroxyesters, dialkyl peroxides, hydroperoxides or peroxyketals. Examples of preferred organic peroxides are cumene hydroperoxide, benzoyl peroxide or t-butyl hydroperoxide.
  • the oxidizing compound preferably is kept in a separate container from the cyclic hydrazide as mixing the solutions may reduce the shelf life of the hydrazide.
  • the oxidizing compound may be organic peroxides, inorganic peroxides, or mixtures of the two.
  • the peroxide compound may be added as a dry solid, for example,
  • peroxycarbonate granules but the preferred embodiment is a liquid containing a trace metal ion chelator.
  • This liquid may be either poured or sprayed into the receptacle for receiving the human excrement.
  • the inorganic peroxide, organic peroxide or mixture of the two may be added to the water in the toilet bowl either before or after defecation or urination. If the oxidizer is added before the specimen, then following defecation or urination, additional peroxide compound may be applied to the surface of the water.
  • a preferred means of application is for the reagent to be poured into the commode water.
  • the simplest oxidizing solution that performs well in the novel assay of this invention is hydrogen peroxide. Over-the-counter three percent (3%) hydrogen peroxide functions well as an oxidizer.
  • a trace metal chelator must be added to prevent false-positive reactions in the toilet water.
  • Hydrogen peroxide must not be used with EDTA in the context of a luminol assay for the detection of blood.
  • sodium peroxycarbonate and sodium perborate can be used in conjunction with EDTA.
  • An acceptable chelator should have a pH in the range of 4 -7, preferably near neutral, and be compatible with the oxidizer component of the oxidizing reagent.
  • a simple test such as follows, may be utilized.
  • the proposed chelator should be mixed with the oxidizer of choice and the mixture capped.
  • the oxidizer levels are determined using any suitable method for detection of the oxidizer.
  • levels are determined using any suitable method for the determination of hydrogen peroxide.
  • Such methods may include, inter alia, titration, spectrophotometry, fluorescence, chemiluminescence, electrochemical or any other method suitable for detection of hydrogen peroxide.
  • There are also commercial kits available for hydrogen peroxide detection which may be employed. If no substantial change has occurred in hydrogen peroxide levels, the chelator may be used in the method of the present invention.
  • a commercially available chelator compatible with hydrogen peroxide is a phosphoric acid-based chelator sold under the tradename DEQUEST FS 0520 as a solution with excellent transition metals chelation properties. (Solutia, Inc., St. Louis, Missouri). This product has a pH of 5.0 and is capable of binding the trace metals in water. The preferred concentration of Dequest FS 0520 is one hundred cc per liter of 3% hydrogen peroxide.
  • a reagent comprising a chemiluminescent agent is added to the receptacle containing the specimen.
  • the chemiluminescent agent is selected from the purified sodium or potassium salts of luminol (5-amino-2,3-dihydro 1 ,4- phthalazinedione; o-amino-phthalyhydrazide) and/or the sodium or potassium salts of benzo (ghi) perylene-1 ,2-dicarboxylic acid.
  • chemiluminescence produced by the occult blood may be detected by the naked eye in the dark without the aid of instrumentation.
  • the same chemiluminescent reagent is also used in the negative and positive controls in steps 1 B and 2C.
  • chemiluminescence may also be achieved with cyclic or acyclic hydrazides.
  • cyclic hydrazides analogs with substituents on the nonheterocyclic ring are easily prepared, but any substitution of the heterocyclic ring renders the compounds non-chemiluminescent.
  • Cyclic hydrazide compounds which are up to 150% more efficient in chemiluminescence than luminol, such as benzo (ghi) perylene-1 ,2-dicarboxylic acid hydrazide, can be prepared according to the method of Wei and White. Tetrahdron Letters: Volume 39: 3559 (1971 ) by C. C. Wei and E. H.
  • Non-interfering substances which can augment the chemiluminescence are contemplated, but the sensitivity should be sufficient as to not require augmenting the chemiluminescence of the luminol.
  • An example of a non-interfering substance would be fluorscein.
  • Chemiluminescence is the emission of light (424 nm wave length) from chemical reactions at ordinary temperatures.
  • One example of chemiluminescence is the oxidation of luminol (5-amino-2,3-dihydro 1 ,4-phthalazinedione; o-amino- phthalylhydrazide), to produce an intense neon-like blue light.
  • Luminol exhibits an intense neon-like blue chemiluminescence by oxidation in an alkaline solution.
  • Luminol reacts with animal blood as well as with human blood.
  • the advantage of luminol for the detection of blood is its extreme sensitivity. Hematin can be detected in a dilution of 1 :1 ,000,000 or 10 "4 micrograms of crystalline hemoglobin. The duration of the luminescence depends upon the amount of blood present.
  • luminol is used in forensic science for the detection of blood at crime scenes. Analytical chemists have also used luminol for the detection of trace amounts of iron, cobalt, copper and chromium (III). Free radical generation from erythrocytes or whole blood has been monitored continuously by luminol-amplified chemiluminescence.
  • reaction path shown in FIG. 2 is proposed as an explanation for the chemiluminescence of luminol at an alkaline pH.
  • the ambient light is eliminated, and the specimen may be observed with the unaided eye for the presence or absence of a neon-like chemiluminescent blue light.
  • chemiluminescence production of a blue light is indicative of blood.
  • the light emission produced with the purified luminol reagent according to the method of the invention is as much as one hundred fold greater than the light emission of unpurified luminol.
  • the reaction of one drop of blood in a flask containing 1800 cc of water demonstrates that purified Luminol can detect 5 nanograms of hemoglobin per ml_.
  • the reaction product can be easily seen with normal vision without the aid of photodiodes or photomultipliers
  • An appropriate purity of luminol salt must be utilized. If the luminol starting material has contaminants, then a procedure for purifying luminol must be used. A procedure for the purification of luminol is provided herein. Other purification methods may also be effective. The level of purity can be tested by determining if a known sample comprising a drop of blood oxidized with 3 ml_ of hydrogen peroxide can be detected as stated above with addition of 5 ml_ of an aqueous solution of sodium luminol (2 mg/mL sodium luminol; 75 mg/mL sodium hydroxide) to said oxidized blood with the chemiluminescence visible to the unaided eye.
  • the luminol product has a starting purity of >99% with crystals that are cream to white in color, it may be used in the method of invention without further purification providing the chemiluminescence produced is at least 75% of the glow intensity seen with the purified luminol.
  • Luminol crystals with >99% purity demonstrating acceptable chemiluminescence as described herein may be acquired from Gold Biotechnology of St. Louis, MO.
  • the luminol does not perform to the standard indicated above, it may be purified by the following method.
  • crude luminol is first converted to sodium luminol.
  • Crude luminol is dissolved to near saturation in 5% weight/volume room temperature sodium hydroxide (i.e. 100 mg/mL), suction filtered through a 0.22 micron membrane (Millipore), and then crystallized at 0°C for four hours.
  • the sodium luminol precipitates out leaving a supernatant that is solid black.
  • the precipitated crystals are filtered on a Whatman GFA disk, then dissolved in the minimum volume of 5% sodium hydroxide and re-crystallized at 0°C for 18 hours.
  • An alternative method of purifying luminol is as follows. One gram of crude luminol is added to 50 mL of deionized water. Seven mL of 1 Molar sodium hydroxide is added to the solution, followed by the drop-wise addition of 1 mL of concentrated hydrochloric acid (37%). The hydrochloric acid brings the pH back to 7.00. During the addition of hydrochloric acid a white precipitate appears. This is purified luminol. The supernatant is removed by aspiration and the precipitate washed with deionized water. The white precipitate is warmed in an oven to evaporate any remaining water. Chemiluminescent Reagent - Preparation of Preferred Luminol Reagent
  • a chemiluminescent reagent comprising a basic solution of purified sodium luminol is prepared by solubilizing purified sodium luminol in 75 mg/mL of sodium hydroxide to make a solution with a concentration of from about 0.01 to 5 mg/mL sodium luminol.
  • the preferred concentration of the chemiluminescent reagent is about 2.0 mg/mL sodium luminol since above this concentration there is only a slight increase in light output.
  • the near zenith of chemiluminescence of purified sodium luminol occurs with a concentration of 2 mg/mL.
  • the concentration of sodium hydroxide used in the luminol solution should be sufficient to raise the water in the testing system to a pH of greater than 10, and greater or lesser than 75 mg/mL may be required to achieve that pH.
  • the most preferred concentration of sodium hydroxide is 75 mg/mL, which is adequate to raise the pH of the water to the optimum pH of 12.4.
  • FIG. 3 shows chemiluminescence vs. time of several luminol concentrations at specified pH values.
  • the solution can be Q.S. to pH 12.4 with concentrated hydrochloric acid, per Table II below.
  • the average volume of water in a toilet bowl is about 1800 mL.
  • Addition of one ounce (30 mL) of the chemiluminescent reagent containing 2 mg/mL sodium luminol in 75 mg/mL of sodium hydroxide will raise the pH of the toilet water to about 12.4. This is necessary to achieve optimal light output allowing the chemiluminescence to be seen with the naked eye in a darkened room without the need of instrumentation.
  • the optimal method for detecting occult blood is to perform the test with a pH greater than 10.0, and preferably at a pH of near 12. 4. At this pH, most, if not all, plant peroxidases will be inactivated allowing maximal chemiluminescence to occur and elimination of the false positives caused by plant peroxidases seen with previous FOBT performed at pH 9.0 or less. By raising the pH to greater than 12, hydrogen peroxide is converted to perhydroxyl ions. This is the preferred species of hydrogen peroxide for the oxidation of luminol.
  • the pH requirement to reduce or eliminate false-positives due to dietary peroxidases, i.e. > 10 and preferably at pH 12.4, is critical with sodium luminol, which point has not been recognized prior to this invention.
  • Tables I and II below provide preferred ingredient ratios for the sodium luminol reagent.
  • An alternative preferred chemiluminescent reagent is disodium benzyl (ghi) perylene hydrazide 1 ,2 dicarboxylic acid.
  • Formulations for the alternative reagent of the present invention are given in Tables III and IV below. Table III
  • a solution of 5% hydrogen peroxide was poured into a two-ounce bottle fitted with a Calmar ® SSA 0.025 spray nozzle (Calamar, Lee's Summit, MO).
  • the hydrogen peroxide solution was prepared by diluting a 50% hydrogen peroxide (Food Grade - Solvay Interox) solution one to ten with deionized water.
  • An aqueous sodium luminol solution (2.0 mg/mL sodium luminol in 2 Molar sodium hydroxide) was also poured into a two ounce bottle fitted with a Calmar ® SSA 0.025 spray nozzle.
  • Turnips, radishes, carrots, apples, horseradish sauce, cantaloupe, watermelon, and celery were purchased from a grocery store. Each vegetable was ground into a slurry with a blender. For each vegetable tested, two ounces of hydrogen peroxide was poured into the commode water, and then the vegetable slurry was added. An aqueous solution of sodium luminol (1 mg/mL sodium luminol; 14 mg/mL sodium hydroxide) was sprayed into the commode water. The bathroom light was then turned off and the water was observed for the blue glow of chemiluminescence.
  • Commode water will test negative for chemiluminescence when the oxidizing solution containing chelators and then the sodium luminol/sodium hydroxide solution are sprayed on the water's surface unless the water has been contaminated by toilet bowl cleaners. It has been observed that some commodes will produce a brilliant, glowing blue light during drainage of the commode water during flushing. This is thought to occur due to trace metal contamination from the toilet bowl tank.
  • Commodes that have been disinfected with hypochlorites may produce a false positive reaction.
  • the reaction with hypochlorites is intense, but brief, lasting less than five seconds. Therefore, false positives from toilet bowl cleaners containing
  • hypochlorites can be determined by the distribution, intensity, and length of reaction.
  • EXAMPLE 8 DETECTION OF POLYPS IN A 70 YEAR OLD MAN
  • guaiac-based test cards (HEMOCCULT®) were obtained and were also numbered consecutively one through five. One cc of fluid from each cup was used to thoroughly soak the cards. These were allowed to air dry. The developer was then used per the package directions.
  • the assay of the novel chemiluminescent invention was performed by adding 3 cc of 3% hydrogen peroxide to each cup and mixing. In a dark room, 0.6 cc of purified sodium luminol solution was added to each cup. The results are as follows:
  • the novel invention is at least 200,000 times more sensitive for the detection of hemoglobin than the commercially available guaiac test kits This is important since adenomatous polyps less than 2.5 cm in diameter only lose small amounts of blood, i.e., 1 to 2 cc per day.
  • the commercially available HEMOCCULT® test (Beckman-Colter) requires 10 cc of blood in a fecal specimen in order to provide correct results about 50% of the time.
  • the procedure of the invention can detect hemoglobin at a concentration of 5 nanograms per mL and may be able to detect concentrations in excess of 1 nanograms per mL.
  • the novel FOBT was performed on patients scheduled for elective colonoscopy at the Gl Center in Overland Park, Kansas. Volunteers signed informed consent to perform FOBT with the chemiluminescence test of the present invention as well as a commercially available fecal immunochemistry test kit (FIT) (InSure ® brand, Enterix, Inc, Edison, N.J.). The FDA pre-approved product instructions for experimental use of the chemiluminescence test of the invention, and the patients were provided with these instructions. The patients were required to complete an exit questionnaire to ascertain that they understood the instructions. A diet free of red meat, ascorbic acid, iron supplements and vitamins was recommended for three days prior to any stool testing and through the testing period. Volunteers were instructed follow the procedure for observing the negative control before proceeding with the test samples.
  • FIT fecal immunochemistry test kit
  • the chemiluminescence test kit was positive in eight cases.
  • the commercial fecal immunochemistry test (FIT) kit was negative in all thirty of the volunteers. Results are presented in Tables VI and VII below.
  • chemiluminescence test detected several adenomatous polyps.
  • the smallest adenomatous polyp detected was 6 mm.
  • the other four patients' adenomatous polyps were all 10 mm. All adenomatous polyps were removed with a snare and were free of metaplasia or dysplasia.
  • the chemiluminescence test was positive when polyps were located in the cecum, hepatic flexure, descending colon and rectal area. The chemiluminescence test did not detect any hemorrhage from four patients who had 3 mm sessile polyps.
  • the chemiluminescence test has the ability to detect premalignant lesions in all locations with greater sensitivity than the immunochemical assay and with a high rate of specificity.
  • the 6 mm adenomatous polyp was positive on all three bowel movements. If the chemiluminescent test can detect adenomatous polyps where bleeding from a 1 cm polyp is estimated to be 1 .2 cc per day then it should routinely detect a mucosal adenocarcinoma where the bleeding is estimated to be about 5 cc per day.
  • a standard guaiac test such as Hemoccult®, blood loss of approximately 10 cc per day is needed for the test to correctly detect bleeding in 50% of the samples.
  • Phenalphthalin to detect plant peroxidases in the specimen
  • An additional step may be included in the method of the invention utilizing a chromogenic composition which will change colors in the presence of plant
  • This chromogenic composition would also change colors in the presence of occult blood following the addition of hydrogen peroxide to lyse the red blood cells. The addition of this step would further reduce the incidence of false positives by identifying samples that contain active plant peroxidases, the presence of which could lead to false positive test results.
  • the preferred chromogenic composition comprises an aqueous basic solution of phenolphthalin.
  • the sodium salt of phenolphthalin is colorless in deoxygenated alkaline solution, and is readily oxidized by minute quantities of blood in the presence of hydrogen peroxide to phenolphthalein which gives a deep hot pink color in alkaline solution. If plant peroxidases are present in the fecal specimen, the colorless phenolphthalin will oxidize to phenolphthalein producing a hot pink color which is visible to the unaided eye in ambient room lighting. Following the addition of hydrogen peroxide, occult blood will also produce a bright pink glow. If occult blood or plant peroxidases are not present, the composition will remain clear.
  • the feces is deposited in the toilet bowl receptacle, an alkaline solution containing approximately 2 mg/mL of phenolphthalin in 0.25 moles/L sodium or potassium hydroxide is added to the receptacle containing the specimen, and the receptacle is observed for 30-45 seconds. If a color change to hot pink is visible, plant peroxidases are present in the sample and the test should be discontinued. Appropriate dietary recommendations should be implemented to reduce these peroxidases before retesting.
  • reagents appropriate for a test procedure can be supplied in a kit comprising a container for oxidizing agent, a container of
  • the kit preferably further comprises containers of reagent(s) which test for interfering substances in the toilet bowl and/or peroxidases as described above.

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Abstract

La présente invention concerne un nouveau procédé de détection du sang occulte dans les selles, l'urine, les sucs gastriques et les expectorations. Ledit procédé représente un progrès dans le domaine de la recherche du sang occulte, en particulier sous une forme « d'autotest » simple à utiliser par les non-spécialistes. Le procédé peut être appliqué à un échantillon préalablement déposé dans une zone d'essai, par exemple une cuvette de toilettes, sans qu'il soit nécessaire de manipuler l'échantillon. Le procédé consiste à appliquer un agent oxydant, contenant de préférence un chélateur de métaux, à l'échantillon souhaité, puis à appliquer un composé chimiluminescent ou chimifluorescent. Il faut ensuite éliminer la lumière ambiante pour permettre de détecter la lueur chimiluminescente ou chimifluorescente à l'œil nu. Les composés chimiluminescents ou chimifluorescents de l'invention sont choisis parmi le luminol de sodium ou de potassium purifié et l'hydrazide de benzo(ghi)pérylène-(acide 1,2-dicarboxylique) de sodium ou de potassium purifié, à un pH supérieur à 9,0. L'utilisation de ces sels de sodium ou de potassium d'hydrazides génère, en présence de sang occulte, une chimiluminescence dont l'intensité est visible à l'œil nu, sans matériel de détection.
PCT/US2011/030934 2010-04-01 2011-04-01 Procédé de détection du sang occulte WO2011123776A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013082267A1 (fr) 2011-11-30 2013-06-06 Wheeldon Eric B Appareil et procédé de détection à distance de sang dans les selles et l'urine humaines
US20140147924A1 (en) * 2011-11-30 2014-05-29 Eric B. Wheeldon Apparatus and Method for the Remote Sensing of Blood in Human Feces and Urine
CN105738328A (zh) * 2014-12-10 2016-07-06 胡朝阳 一种家用联网的检测粪便及尿液隐血的系统
JPWO2017038711A1 (ja) * 2015-08-28 2017-08-31 栄研化学株式会社 免疫学的測定用試薬組成物およびその用途
KR20180107277A (ko) * 2016-02-16 2018-10-01 메트리오팜 아게 5-아미노-2,3-디하이드로프탈라진-1,4-디온의 새로운 결정질 형태
JP2019504865A (ja) * 2016-02-16 2019-02-21 メトリオファーム アーゲー ５−アミノ−２，３−ジヒドロフタラジン−１，４−ジオンの結晶形の製造方法
US10575830B2 (en) 2015-02-25 2020-03-03 Outsense Diagnostics Ltd. Bodily emission analysis
CN112858269A (zh) * 2020-12-25 2021-05-28 广东绿康医疗器械有限公司 鼻咽粘膜细胞游离血红素的检测试剂制备方法及使用方法
US11467091B2 (en) 2016-08-30 2022-10-11 Outsense Diagnostics Ltd. Bodily emission analysis

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US4725553A (en) * 1983-09-29 1988-02-16 Helena Laboratories Corporation Test composition for detecting occult blood
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* Cited by examiner, † Cited by third party
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US20140147924A1 (en) * 2011-11-30 2014-05-29 Eric B. Wheeldon Apparatus and Method for the Remote Sensing of Blood in Human Feces and Urine
US8802442B2 (en) * 2011-11-30 2014-08-12 Eric B. Wheeldon Apparatus and method for the remote sensing of blood in human feces and urine
CN104254621A (zh) * 2011-11-30 2014-12-31 埃里克·B·惠尔顿 遥测人粪便和尿液中的血液的装置和方法
EP2785877A4 (fr) * 2011-11-30 2015-06-17 Eric B Wheeldon Appareil et procédé de détection à distance de sang dans les selles et l'urine humaines
AU2012345944B2 (en) * 2011-11-30 2018-11-01 Robert J. Barsotti Apparatus and method for the remote sensing of blood in human feces and urine
WO2013082267A1 (fr) 2011-11-30 2013-06-06 Wheeldon Eric B Appareil et procédé de détection à distance de sang dans les selles et l'urine humaines
CN105738328A (zh) * 2014-12-10 2016-07-06 胡朝阳 一种家用联网的检测粪便及尿液隐血的系统
US10575830B2 (en) 2015-02-25 2020-03-03 Outsense Diagnostics Ltd. Bodily emission analysis
US11786224B2 (en) 2015-02-25 2023-10-17 Outsense Diagnostics Ltd. Bodily emission analysis
US11129599B2 (en) 2015-02-25 2021-09-28 Outsense Diagnostics Ltd. Bodily emission analysis
JPWO2017038711A1 (ja) * 2015-08-28 2017-08-31 栄研化学株式会社 免疫学的測定用試薬組成物およびその用途
CN107923907A (zh) * 2015-08-28 2018-04-17 荣研化学株式会社 免疫学的测定用试剂组合物及其用途
JP2019504865A (ja) * 2016-02-16 2019-02-21 メトリオファーム アーゲー ５−アミノ−２，３−ジヒドロフタラジン−１，４−ジオンの結晶形の製造方法
JP2019509268A (ja) * 2016-02-16 2019-04-04 メトリオファーム アーゲー ５−アミノ−２，３−ジヒドロフタラジン−１，４−ジオンの結晶形の製造方法
JP2022037005A (ja) * 2016-02-16 2022-03-08 メトリオファーム アーゲー ５－アミノ－２，３－ジヒドロフタラジン－１，４－ジオンの結晶形
JP7034079B2 (ja) 2016-02-16 2022-03-11 メトリオファーム アーゲー ５－アミノ－２，３－ジヒドロフタラジン－１，４－ジオンの結晶形の製造方法
KR102535960B1 (ko) * 2016-02-16 2023-05-23 메트리오팜 아게 5-아미노-2,3-디하이드로프탈라진-1,4-디온의 새로운 결정질 형태
JP7331067B2 (ja) 2016-02-16 2023-08-22 メトリオファーム アーゲー ５－アミノ－２，３－ジヒドロフタラジン－１，４－ジオンの結晶形
KR20180107277A (ko) * 2016-02-16 2018-10-01 메트리오팜 아게 5-아미노-2,3-디하이드로프탈라진-1,4-디온의 새로운 결정질 형태
US11467091B2 (en) 2016-08-30 2022-10-11 Outsense Diagnostics Ltd. Bodily emission analysis
US11561181B2 (en) 2016-08-30 2023-01-24 Outsense Diagnostics Ltd. Bodily emission analysis
US11971356B2 (en) 2016-08-30 2024-04-30 Outsense Diagnostics Ltd. Bodily emission analysis
CN112858269A (zh) * 2020-12-25 2021-05-28 广东绿康医疗器械有限公司 鼻咽粘膜细胞游离血红素的检测试剂制备方法及使用方法

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