Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood
Reexamination Certificate
1995-05-12
2002-06-25
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
C436S008000, C436S164000, C436S904000, C422S067000, C435S028000, C435S810000
Reexamination Certificate
active
06410336
ABSTRACT:
This invention relates to the detection of fecal occult blood, and in particular to the use of enhanced developers in the detection of fecal occult blood.
Fecal occult blood may provide a reliable diagnostic indicator of a variety of medical conditions involving gastrointestinal bleeding which may otherwise be difficult to detect, including colorectal cancer. The use of this method is well described in the medical literature. See e.g., Greegor, D. H., Cancer 19; 330-337 (1969) and Hastings, J. B., Amer. J. Surg. 127:228-233 (1974). Tests for fecal occult blood based upon the oxidation of guaiac to form a blue colored product in the presence of hydrogen peroxide and hemoglobin have been described in U.S. Pat. No. 3,996,006, incorporated herein by reference. Such products are sold under the trademarks HEMOCCULT® AND SERACULT®. Briefly, the test involves placing a fecal sample on an absorbent paper coated with guaiac and adding a developer solution containing hydrogen peroxide. If hemoglobin is present, the guaiac is oxidized, turning the paper blue.
It is considered desirable to increase the sensitivity of the test in order to detect excessive blood loss into the bowel at the earliest possible stage. The occurrence of false negatives, the problem of not detecting the presence of blood, is thus detrimental. Attempts have therefore been made to increase the sensitivity of the test. For example, the addition of a drop or two of water to the fecal smear on the guaiac impregnated test paper prior to the addition of the peroxide solution has been used to enhance the test sensitivity. Such procedures frequently give a positive reaction even if there is no pathologically significant amount of blood present in the stool. The occurrence of false positives is also undesirable, since it can lead to unnecessary follow-up medical procedures.
An improved version of this test is described in European Patent Publication No. 0 308 227. Phenols are added to the developer solution to achieve an enhancement of the amount of colored product produced, resulting in a more sensitive assay.
SUMMARY OF THE INVENTION
It has now been found that compounds other than phenols, specifically tertiary and quaternary nitrogen compounds, can be used to enhance the sensitivity of a test for fecal occult blood. It is therefore an object of the present invention to provide an enhanced method for testing of fecal occult blood that makes use of (1) monocyclic nitrogen-containing aromatic heterocyclic compounds such as triazole, pyridine, pyrazine or substituted derivatives thereof; (2) tertiary or quaternary ammonium compounds containing at least one hydroxy alkyl or esterified hydroxy alkyl group or a phenyl or substituted phenyl group attached to the nitrogen or (3) quinoline or a substituted derivative thereof to enhance the sensitivity of the test.
It is a further object of the invention to provide a developer solution containing an enhancer in accordance with the invention together with peroxide in an ethanol water carrier for use in testing for fecal occult blood.
It is still a further object of the invention to provide a kit for testing for fecal occult blood. Such a kit includes an enhanced developer composition containing an enhancer according to the invention and an oxidizable substrate such as guaiac.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, a fecal specimen may be tested for the presence of occult blood by combining the fecal specimen with
(a) an oxidizable substrate that produces a colored product in the presence of peroxide and hemoglobin;
(b) hydrogen peroxide or a peroxide source; and
(c) an enhancer selected from the group consisting of monocyclic nitrogen-containing aromatic heterocyclic compounds or tertiary or quaternary ammonium compounds which enhance the sensitivity of the test. If hemoglobin is present in the fecal sample, the oxidizable substrate is converted to the visibly detectable, colored product.
A preferred oxidizable substrate for use in accordance with the invention is guaiac which is the substrate used in commercially available test kits. Other substrates which are oxidized by peroxide in the presence of hemoglobin to produce a colored product that can be visually detected in the presence of fecal material may also be used. Suitable alternative materials include 3,3′,5,5-tetra-methylbenzidine (see U.S. Pat. No. 4,562,043 incorporated herein by reference), guaiaconic acid A and other leuco dyes that are oxidized in the presence of peroxide and hemoglobin (see U.S. Pat. Nos. 4,219,336 and 4,971,914 incorporated herein by reference).
A simple and convenient way to conduct the test is to place the oxidizable substrate on a solid support. A preferred solid support is an absorptive paper which has been impregnated with the oxidizable substrate. The oxidizable substrate needs to be present in amounts sufficient to generate a detectable amount of colored products, generally above 0.5 cm/1000 in
2
. In the coated paper used in commercial kits today, this amounts to about 0.75 gm/1000 in
2
. Using a micropipet, a small (e.g. 0.03 ml) aliquot of the fecal specimen is placed on the support., One or two drops of developer solution are then added, and the extent of color development, if any, is observed.
This developer solution contains hydrogen peroxide or a source of peroxide such as cumene hydroperoxide and the enhancer in an ethanol/water carrier. The developer solutions of the invention generally contain 4 to 6% by weight of hydrogen peroxide, 0.2 to 4% by weight of the enhancer, and 80 to 90% ethanol with the balance being water. Preferably, the enhancer will be present in amounts of from 0.3 to 1.5% by weight, the exact amount depending on the specific enhancer being used. Unless limited by solubility considerations, higher enhancer concentrations can also be used but it will be appreciated that the resulting increase in sensitivity may lead to the detection of low levels of occult blood that are not medically significant, i.e., false positives.
Enhancers useful in the present invention include monocyclic nitrogen-containing aromatic heterocyclic compounds such as triazole, pyridine, pyrazine and substituted derivatives thereof, including 4-benzyl pyridine, 2-methoxy pyridine, 4-(p-nitrobenzyl) pyridine, and pyrazine carboxylic acid. Other enhancers in accordance with the invention are tertiary or quaternary amines having at least one hydroxy alkyl or esterified hydroxy alkyl group attached to the nitrogen. Examples of such enhancers include diethyl ethanolamine, ethyl diethanolamine and triethanolamine, acetylcholine chloride and &bgr;-methyl acetylcholine chloride. A further class of enhancers in accordance with the present invention are tertiary and quaternary amines having a phenyl group attached to the nitrogen, e.g. dimethyl aniline. Quinoline and derivatives thereof such as alpha-hydroxy quinoline were also found to be effective enhancers.
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Ostrolenk Faber Gerb & Soffen, LLP
Propper Manufacturing Co., Ltd.
Wallenhorst Maureen M.
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