WO1987001460A1

WO1987001460A1 - Test device for determination of blood in faeces

Info

Publication number: WO1987001460A1
Application number: PCT/NO1986/000060
Authority: WO
Inventors: Terje Silsand
Original assignee: Terje Silsand
Priority date: 1985-08-30
Filing date: 1986-08-26
Publication date: 1987-03-12
Also published as: NO157437B; DK180587D0; NO853413L; DK180587A; AU6286186A; EP0235241A1; NO157437C

Abstract

Faeces is applied on an absorbing paper impregnated with a solid peroxide-giving compound, here sodiumperborate. To the faeces sample is added a chromogen, 3,3',5,5'-tetramethylbenzidine, dissolved in acetic acid, ethanol and water and stabilized by adding histidine. When blood is present in the faeces sample blue-bluishgreen colour developes on and at the edge of the faeces sample. Coulor development can occur within 5-10 seconds when the sample contains 1 ml blood pr. 100 g faeces. The analytical system thus employs only one single reagent when the assay is performed because the system's peroxide-giving part is impregnated on the same carrier on to which the faeces is applied.

Description

TEST DEVICE FOR DETERMINATION OF BLOOD IN FAECES.

The invention concerns a test device for determin¬ ation of blood in faeces. Common for all systems are that blood act as a catalyst in a reaction in which a colour¬ less chromogen is transformed to a strongly coloured com- pound when hydrogenperoxide (H„0_) is added to the systems, or is generated in the systems. A development of colour is interprated as positive; that blood can be present in faeces. Common for all systems is that heme will dissociate from hemoglobin in an acid solution and act as a pseudoperoxidase.

The systems currently in use are, with few exceptions, constructed in the following way: Faeces is applied on a piece of paper. The paper may be impregnated with a chromogen (guaiacol is mostly used) or not impreg- nated. In the systems with impregnated paper H O , diss¬ olved in an alcohol-containing solvent, is applied to the paper. In the systems where the paper is not impregnated with a chromogen a solution containing a chromogen (e.g. 3 , 3' , 5 , 5' -tetramethylbenzidine (TMB) dissolved in a strongly acid alcohol-containing solvent) is added to the paper followed by adding a solution of H„0_. Common for the systems are that in the presence of blood a colour development occurs on and at the edge of the faeces.

^• The hitherto described systems are constructed n such a way that the paper, with or without chromogen at the same time is carrier for the faeces. A carrier is here defined as a place where the faeces is stored until assay is performed. The systems are primarily constructed for the purpose that the patient himself applies faeces at home for later on to bring the paper to the physician or laboratory. This require that no reaction must take place between blood in the faeces and components on the paper. At hospitals the assay is mostly performed shortly after applying the sample on the paper. The systems described hitherto where the paper is impregnated with chromogen (guaiacol) suffer from some disadvantages. One of these systems is produced by Lab- systems, Helsinki, Finland, and is sold under the trade-

to* mark "Fecatwin Sensitive". The manufacturer recommends to store the faeces on the paper for at least 2 hours before performing the assay, and claims that 72 hours storage ensures best result. The rate of positive findings is far higher when the samples are stored for 72 hours, which is confirmed by Dybdahl. (Dybdahl J.H., Studies on Occult Faecal Blood Loss, Thesis, Det medisinske fakultet, Uni- versitetet i Oslo, 1983). It is an obvious disadvantage that a test's sensitivity is dependent on the storage time of the sample, and the disadvantage is very serious when the assay may give false negative result when assay is performed shortly after applying the sample. Another of the systems with chromogeπ-impregnated paper is "Hemoccult" (U.S. Patent No 3,996,006). One disadvan- tage in this system is that faeces cannot be stored for more than 12 days before assay must be performed. Chromogen-impregnated paper may give false positive react¬ ion when peroxidases from unboiled vegetables are present in faeces. Such peroxidases may, in sufficiently high concentrations, oxidase the chromogen to a coloured com¬ pound in the absence of peroxide. It has been shown ( own unpublished results) that guaiacol-based systems gave positive reaction in the presence of horse radish perox- idase without adding peroxide. Such unwanted reactions cannot occur when faeces is stored on paper impregnated with peroxide in the absence of chromogen. Test devices where carrier is chromogen-impregnated may also autooxidase, and the production process makes it simpler to impregnate with peroxide than chromogen. The guaiacol-based systems cannot be stored at low temperature, + 4 C or lower. Distribution may therefore be a problem in the cold season.

By making a system where the carrier, as defined here, is peroxide-impregnated and not chromogen-impregnated one avoids the disadvantages described for the chromogen- based systems. The new system keeps chromogen separated from peroxide (and applied faeces) until assay is performed. The system allows performance of assay immediately after applying faeces, e.g. in a hospital. The system also allows the patient to apply the sample in his home for later on to bring the paper to physician or laboratory for assay. Own experiments show that applied samples can be stored for at least 10 weeks at room temperature before assay is performed. It will be understood that physician or laboratory shall perform the assay. Law regulations in very many coπtries makes it inecoπomical for the patient to perform the assay in his home .

Use of solid peroxide compound is known (European Patent Application nr. 93595), and then combined with a chromogen (guaiacol) to form a single reagent package. The described reagent package seems to be constructed for depo- siting it in a toilet bowl containing faeces. Hemoglobin dissolved in the water will catalyze the reaction between chromogen and peroxide. Faeces cannot be stored on the reagent package for subsequent assay as described for the systems with chromogen-impregnated paper, and as described for the new system. The reason is that water in faeces may dissolve chromogen and peroxide causing the catalytical reaction to start.

The new system thus differs from European Patent Application nr. 93595 by keeping peroxide and chromogen separate, and that the peroxide-impregnated paper first serves as carrier (storage place) for faeces, and later as analytical part of the system when chromogen is applied. European Patent Application 93595 is also based on a reaction in an aqueous system, while the new system employs mainly ethanol and acetic acid and very little ater. It can here be mentioned that by increasing the amount of water in test systems for determination of blood in faeces the risk increases for obtaining false positive reactions due to the presence of peroxidases from vegetables in faeces. For . practica 1 purpose the new system use absorbing paper impregnated with a solid peroxide-containing compound, here sodiumperborate . In principle this is simple, but not without problems. Experiments with differnt types of paper show that sodiumperborate is very unstable when impregnated on certain types of paper in the meaning that after a short period of time the reaction between blood and chromogen is not obtained. For those types of paper which show bad stab-^' ility for sodiumperborate the problem can be solved by dissolving sodiumperborate in an aqueous solution containing organic acid, e.g. acetic acid, or a mixture of acetic acid and citric acid. By using organic acid one obtains that

10 higher concentrations of sodiumperborate can be prepared compared to an aqueous solution not .containing organic acid. The impregnation of the paper can be done in one operation using a concentrated solution of sodiumperborate containing organic acid instead of repeated impregna ions in less

15^'* concentrated solutions, thus simplifying the impregnation. To ensure optimal stability of the paper impregnated with sodiumperborate the concentration of sodiumpe borate and acid must be balanced, avoiding too high concentration of acid. The ratio can be exemplified: The concentration of

20 acetic acid should not exceed 0.5 N when the concentration of sodiumperbora e is 100 g/1.

The paper impregnated with sufficient amounts of sodiumperborate shows long stability as part of the reagents^' used for determination of blood in faeces; It can

25. be stored at least 12 months when refrigerated. To obtain such effect it is sufficient to impregnate the paper once in a 100 g/1 sodiumperborate solution containing organic acid. An alternative way to impregnate the paper is to use less concentrated solutions of sodiumperborate without

30 organic acid, with repeated impregnations and drying. This alternative can be used using paper types showing sufficient stability of sodiumperborate in the absence of organic acid.

35^r; Description of the test device.

The test device is composed of two reagents, here¬ after designated Reagent A and Reagent B. Reagent A is an absorbing paper impregnated with a solid peroxide-contain¬ ing compound, here sodiumperborate. The paper serves both as a carrier for the system's peroxide and as an applicat¬ ion paper (storage place) for the faeces sample. Reagent B contains 3 , 3 ' , 5 , 5 ' -tetramethy lbenzidine (TMB) dissolved in ethy la lcoho1 , acetic acid and water. Histidine is added to the solution to stabilize TMB. (R.M. Jaffe and W . Zierdt. J Lab Clin Med. (1979) 93, 879-886).

Reagent B is added to the faeces sample applied on Reagent "A . In the presence of blood blue-bluishgreen colour devel- opes on and at the edge of the faeces sample. The reaction between blood, chromogen and peroxide is very fast, e.g. a sample containing approximately 1 ml blood (with hemoglobin concentration approximately 15 g/100 ml) pr. 100 g faeces will give a distinct colour within 5-10 seconds. The test device is described in its simplest form, and any variation in packaging technique, especially for covering the faeces sample, will not change the principle of the test dev ice .

Table 1. Composition of the test device

Reagent A has the following prefered composition: A piece of filter paper is dipped in a solution of sodiumperborate, - 100 g/1, containing acetic acid up to 0.5 N. A mixture of acetic acid and citric acid can be used instead of acetic acid alone: Normality acetic acid :Normality citric acid= 3:1, with maximum acid concentration 0.5 N. Alternatively, the paper can be impregnated in less concentrated sαdium- perborate solutions without organic acids, with repeated impregnations and drying.

Reagent B has the following prefered composition: 3 , 3' , 5, 5' -tetramethy lbenzidine 2 g/1 solution

Ethylalcohol 500 ml/1 solution - Acetic acid 350 ml/l solution

Water 150 ml/1 solution

Histidine 4 g/1 solution

Claims

P A T E N T C L A I M

1. Test device for determination of blood in faeces composed of two separated reagents; a filter paper 5 impregnated with solid peroxide, her by dipping the paper in a solution of sodiumperborate with concentration equivalent to 60-120 g/1, and a liquid reagent containing a chromogen ( 3 , 3 ' , , 5 ' -tetramethylbenzidine (1-3 g/1) dissolved in ethano 1 : acetic acid:water= 5:3.5:1.5, containing

10 histidine (4g/l), c h a r a c t e r i z e d b y that peroxide and chromogen are separated, and that the faeces sample is applied to the peroxide-impregnated filter paper and can be stored there until the assay is performed by adding the chromogen to the filter paper containing faeces

15 and peroxide whereby colour developes in the presence of blood .

20

2.5

30

35