FI76217B - PROVTAGNINGSANORDNING FOER UPPSAMLING AV ETT AVFOERINGS-TESTPROV. - Google Patents

Description

This invention relates to a sampling device for collecting and processing a hemoglobin -fyrine and measuring its fluorescence, preferably as described in the parent application of this application.

Various rapid screening tests to determine elevated hemoglobin values in biological agents such as feces are commonly available. These tests are used throughout medicine as the primary test for detecting intestinal tumors. It is estimated that more than one million such tests are performed each year in the United States for this purpose. Despite the fact that these tests do not give quantitative results and that errors in test results are extremely costly, both personally and financially, and despite the fact that publicly available tests give a considerable number of false positive and negative results, they are constantly used because there is no alternative. .

Commonly available screening tests for the detection of hemoglobin in faeces do not involve the conversion of the hemoglobin to porphyrin and the measurement of its fluorescence; rather, the commonly used tests are indirect tests based on hemoglobin peroxidase-type (pseudoperoxidase) activity. In these tests, colorless leuco dyes become colored in the presence of hemoglobin upon addition of the appropriate peroxide. However, such a test has several limitations. First, due to various factors, such as the non-specificity of the test and the fact that the reaction is usually interfered with or affected by substances such as iron, ascorbic acid or changes in the hemoglobin molecule, false positive and false negative results are significantly obtained. Second, the interpretation of commercially available tests is often unclear because test results are reported only as "positive" or "negative." In addition, due to differences in the sensitivity of different tests, the amount of feces in the samples tested can easily vary by a factor of the order of 20 or more. These factors, as well as the non-specificities and differences in personal interpretation of color development presented above, all limit the usefulness of these tests. Due to these limitations, the test for latent blood is the few remaining non-quantitative tests in clinical and laboratory medicine.

Although no quantitative tests are already available to detect hemoglobin in faeces, urine, or gastric fluid, including the conversion of heme to protoporphyrin, various studies have been performed in the past to address this change. For example, a study by G.R. Morrison, Fluorometric Microdetermination of Heme Protein (Anal. Chem., 37: 1124-1126, 1965) discloses a method in which heme was converted to porphyrin using oxalic acid to measure hemiprotein from animal tissues, followed by fluorescence measurement. However, this method was ineffective for the quantitative determination of hemoglobin above certain concentrations. Under the conditions presented by Morrison, feces with elevated hemoglobin levels should be diluted several thousand times. Such infinite dilution is not suitable for large-scale screening tests.

Thus, there is a need for a quantitative test method for the determination of hemoglobin in biological agents, 3 7621 7 such as faeces, urine and gastric fluid, which test eliminates or significantly reduces the number of false positive and false negative results and is well suited for mass screening test purposes.

The invention thus relates to a sampling device for collecting a faecal test sample of a desired and predetermined size, for the quantitative determination of a test sample comprising an elongate sampling body, a tubular part and characterized by a closed portion having a closed portion the shape of the outer surface and having at least one grooved portion in the outer surface, and that the tubular portion consists of a hollow core open at both ends, through which the size of the entire opening and the shape of the inner portion approximately correspond to the diameter and shape of the sample collection portion to remove excess feces from it.

Compared to the prior art, the device of the present invention eliminates false positive and false negative results and is particularly suitable for pulp screening use. The device according to the invention for collecting and preparing a suitable test stool sample has been developed for use in either a laboratory or an automated method.

In the assay, non-fluorescent hemoglobin is quantitatively converted to fluorescent porphyrin at all hemoglobin concentrations tested. This change occurs when the hemi compounds in the sample are heated using an appropriate concentration-modifying reaction mixture consisting of a reducing acid such as oxalic acid and a reducing salt such as Ferrooxalate. The concentration of porphyrin formed in this method is determined by fluorescence measurement. Such fluorescence is performed on the reaction product in a solid system or after suitable extraction or dilution of the reaction product in a liquid system.

The automated screening method is based on the same principles, for the quantitative determination of fluorescence in a solid system. In the automated process, the reaction mixture may also be gel or pasty in consistency at room temperature but liquefies upon heating.

With the sampling device according to the invention, a test sample of a certain size is collected from the faeces to be tested. The concentration of hemoglobin in such a sample is preferably determined in an apparatus with a plurality of reaction chambers, each containing a suitable amount of either a reaction mixture or a non-reactive composition. The sampling device is then constructed of substances which are solid at ambient temperature but which liquefy and mix with the substance in the various reaction chambers when exposed to the temperatures at which the conversion reaction is carried out. In a preferred embodiment, the composition of the sampling device is similar to that of oxalic acid: the composition of the carrier used for the ferrooxalate and citric acid samples.

The sampling device according to the present invention is shown in Figures 1-6 as follows:

Figure 1 is a cross-section of a sampling device for collecting a stool sample.

Figure 2 is a cross-sectional view of a sampling device for collecting a stool sample with the sleeve in the up position.

Figure 3 is a plan view of a sampling device for collecting a stool sample.

Figure 4 is a cross-sectional view of a sampling device for collecting a stool sample, taken along section line 4-4 of Figure 3.

Figure 5 is a cross-sectional view of a sampling device for collecting a stool sample, taken along section line 5-5 of Figure 3.

Figure 6 is a plan view of the lower end of the sample collection device showing the collected sample in its grooves.

7621 7 5

Figures 7 to 10 show an apparatus in which the hemoglobin determination of a sample collected by a sampling device according to the invention can advantageously be performed.

Figure 7 shows a drawing of an apparatus or reaction vessel in which a test reaction is performed.

Figure 8 shows one reaction chamber from the inside with the reaction mixture and the test sample placed therein.

Figure 9 shows a drawing of the sample reaction vessel to be tested and the envelope in which it is mailed.

Figure 10 is a schematic diagram showing automatic processing equipment.

The sampling device according to the invention is illustrated in Figures 1-5. It can be seen from these figures that this device, generally indicated by reference numeral 10, comprises an elongate, substantially cylindrical sampling rod 12 and an upper, substantially cylindrical portion 11 having a diameter greater than that of the rod 12. The rod portion 12 is integrally joined to the portion 11 at the shoulder 13. The sampling rod 12 has a breaking point 15 by means of which the rod 12 can be cut into two parts after the test sample has been taken. The lower part of the rod 12 has a plurality of grooves 16 running along the outer circumference of the rod 12 to collect the sample to be tested.

The device 10 also includes a substantially cylindrical tubular portion or sleeve 14 having an inner diameter approximately equal to the outer diameter of the rod portion 12, with the sleeve 14 sliding over the rod 12 with only a small gap therebetween. This particular sampling rod 12 is suitable for collecting a faecal sample of a predetermined size. When sampling by means of the sampling device 10, the sleeve 14 is raised so that its upper edge contacts the outer part 13, as shown in Figure 2. The lower part of the rod 12 is then immersed in the faeces to a point immediately below the lower part of the sleeve 14. During immersion, the rod 12 is rotated 6 7621 7 once or twice to ensure that the required amount of feces penetrates the grooves 16. The rod 12 is then removed from the faeces and the sleeve 14 is lowered along the entire rod portion 12. Since there is only a small clearance between the outer diameter of the rod 12 and the inner diameter of the sleeve 4, practically all the faeces from the rod 12 leave, except the one remaining in the grooves 16.

After the sleeve 14 is removed and discarded, the rod 12 is broken at the breaking point. The lower part of the rod 12 shown in Figure 6 is then placed in a reaction chamber, the structure of which is shown in Figures 7 and 8. As shown in Figure 7, the apparatus 18 has a plurality of reaction chambers 19a, 19b, 20a and 20b. These reaction chambers are provided with a lid or cover 21 which is hinged to the main vessel. Each reaction chamber is also provided with a transparent window 22 that is transparent enough to transmit light at wavelengths ranging from about 350 nm to 700 nm. This makes it possible to measure the fluorescence of the substance in the chamber directly through the windows 22.

As described in more detail below, each of the chambers 19a, 19b, 20a and 20b is partially filled with a reaction mixture of a reducing acid and a reducing salt such as oxalic acid: ferrooxalate reaction mixture, or a blank test mixture such as citric acid control. In a preferred embodiment, chambers 19a and 20a are provided with a reaction mixture, such as an oxalic acid and ferrooxalate composition, while chambers 19b and 20b contain a control mixture, such as a citric acid composition. The mixtures of the reaction chambers are to be contacted with the test sample. The device 18, which includes the reaction chambers, can be constructed of many different materials, but preferably it should be constructed of any available plastic having the following properties. First, the reaction chambers, or at least the windows 22, must be sufficiently transparent to transmit light from a wavelength of about 350 nm to 700 7 7621 7 nm for fluorescence measurement. Second, the substance must not react with the mixture in the reaction chamber or in any other way significantly interfere with the measurement. Third, the substance must be optically and chemically stable when heated to 120 ° in an autoclave or oven.

The next step in the assay is to convert the hemoglobin portion of the test sample to metal-free porphyrin. This is done by adding a sampling device or a measured volume of homogenized, diluted test substance to the appropriate mixture. In a preferred embodiment of the automated method, the test samples prepared are combined with a reducing acid and a reducing salt, namely oxalic acid and ferrooxalate, in reaction chambers 19a and 20a. Each of these chambers 19a and 20a contains a composition of 2 molar oxalic acid and 1/18 (0.05) molar Ferrooxalate together with a suitable carrier. Concentrations of about 0.01 to 0.2 molar Ferrooxalate give satisfactory results. The carrier preferably consists of a mixture of polyethylene glycols of different molecular weights. Although the consistency of the carrier may be different or it may have different properties, i.e. it may be a solid or powder such as glass fiber, cellulose powder or metal salt impregnated with oxalic or citric acid, liquid, etc., the carrier has preferably a gel-like, pasty or non-flowable consistency at room temperature to prevent spillage or leakage or run-off from the reaction chambers of the device 18. Preferably, the carrier liquefies at temperatures above 100 ° C. The amount of the reaction mixture to be introduced into each of the reaction chambers 19a and 20a is sufficient to react with the amount of fecal sample collected by the above-described sampling device.

The reaction chambers 19b and 20b in the apparatus 8 7621 7 or reaction vessel 18 shown in Figure 7 contain the same support, but instead of an oxalic acid: ero-oxalate mixture, they contain a non-reducing acid. In a preferred method, this non-reducing acid is 1.5 molar citric acid. Thus, chambers 19b and 20b are control blank chambers used only to measure the fluorescence naturally present in a test sample.

In a preferred embodiment, each of the chambers 19a, 19b, 20a and 20b is filled to about 50-60% of its volume with either an oxalic acid: ferrooxalate system or a citric acid system. When the sampling rod 12 (Figure 6) is added together with the collected test sample, the chamber fills to about 65-75% of its volume, as shown in Figure 8.

The total number of chambers in each device may vary, however, each device preferably includes matching pairs of chambers to collect replicates. One of the samples is used in the reaction chamber where the heme is converted to porphyrin, while the other sample is used in the blank control chamber. Thus, in the device shown in Figure 7, with a total of four chambers, four samples from a single stool sample or two samples from each of the two stool samples can be tested. These samples can be taken by the patient using the device shown in Figures 1-6 and then mailed in a suitable envelope 24 (Figure 9) to the central laboratory for sample processing and analysis.

A carrier comprising a reaction mixture of a reducing acid and a reducing salt and a blank of an inert acid may consist of many different compositions, although the process may be carried out in a liquid, solid or many other systems, the carrier preferably being gel-like, pasty or non-room temperature. consistency and liquefies at temperatures above 100 eC. The carrier composition should also have low or negligible fluorescence so as not to interfere with the fluorescence measurement of the test sample and, in addition, should be stable under all reaction conditions. The use of a semi-solid gel-type carrier is particularly advantageous when the test is used by relatively untrained individuals and when the sample is to be mailed. In the absence of these limitations, it may be advantageous to add feces and fecal sampler directly to aqueous solutions of oxalic acid: ferrooxalate and citric acid, as these aqueous solutions are less non-specifically (blank) fluorescent than those containing gel-forming agents. In a preferred embodiment, the carrier consists of a mixture of polyethylene glycols and corresponding high molecular weight compounds such as poly (ethylene oxides). The reaction mixture, which has been found to be acceptable, contains a mixture of 73.8 g of polyethylene glycols, 25.2 g of oxalic acid and 1.0 g of powdered Ferrooxalate. If ferrous sulphate is used, 1.0 g of ferrous sulphate replaces ferrous oxalate. The control blank test mixture, which has been found to be acceptable, contains a mixture of 71.2 g of polyethylene glycol and 28.8 g of citric acid. From the above mixtures, the final concentrations are 2-molar oxalic acid and 1.5-molar citric acid, respectively.

The next step in the automated process is to heat the reaction mixture with the test samples and reactive or non-reactive substances. When heated to the preferred temperature of 120 ° C, the sampling rod 12 containing the stool sample liquefies, thus releasing the feces into the reaction mixture. When heated to 120 ° C, the reaction mixture containing the preferred polyethylene glycol support and either oxalic acid: ferrooxalate reagent or citric acid liquefies or solubilizes. In chambers 19a and 20a, oxalic acid and ferrooxalate react with the hemoglobin in the faecal sample 7621 7 ίο to convert the hemia moiety to fluorescent porphyrin. In chambers 19b and 20b, a quantitatively insignificant reaction takes place. Although the heating may take place at different temperatures and may take different times, in the preferred automated method, the heating temperature in a suitable autoclave or oven is 120 ° C for 90 minutes. The temperature should be high enough to liquefy the reaction mixtures and liquefy the material of the collection rod 12. During the heating step, the reaction mixtures are mixed together with the stool sample, the liquefied sampling rod and the carrier. After the heating step, this uniform mixture is cooled and allowed to solidify again.

In view of how in the above method the sampling rod 12 liquefies and mixes uniformly with the reactive substances, such a rod 12 must have certain properties and characteristics. For example, it should be solid and rigid at temperatures up to 50 ° C and should liquefy at temperatures above 100 eC. Furthermore, it must remain completely miscible with the reactants during cooling and must be stable so that no significant change in its chemical or physical properties occurs when heated or stored below normal ambient temperature for longer. The sampling rod 12 should also be made of a material that is slowly soluble in water and miscible with the chemical reaction mixture in the reaction chamber when both liquefy upon heating. It is also preferred that the sampling rod 12 be made of chemical compounds of the same type as the substances in the reaction mixture, such as polyethylene glycols, although this is not necessary. The sampling rod 12 must also be made of a material that does not interfere with the conversion of hemicomponents to porphyrin or subsequent fluorescence measurement. In view of these requirements, it has been found that a sampling rod 12 made primarily of polyethylene glycol having a molecular weight of about 20,000 in combination with polyethylene oxide having a molecular weight of about 100,000 or other polyethylene glycols can be used to modify the hardness of the material as desired. As discussed above, the sleeve 14, which is actually discarded, can also be made of the same material as the sampling rod 12. However, it is possible to use many other mixtures of these compounds of different molecular weights, or compounds of the same type, as compositions for the preparation of the sampling rod 12.

After the stool sample and the various reaction mixtures in the chambers 19a, 19b, 20a and 20b are heated and then cooled and re-solidified, the fluorescence of each reaction chamber is measured. The general method is then similar to that of a liquid system, except that a light source exciting in liquids is passed through the sample to measure fluorescence at a right angle, whereas in a solid sample, fluorescence is measured from the front surface. In the measurement, the fluorescence of each control blank test sample, reaction chambers 19b and 20b, is subtracted from its corresponding reaction sample, reaction chambers 19a and 20a. The difference between these fluorescence values is then compared with the fluorescence intensity of a known hemoglobin concentration standard and the hemoglobin concentration in the test sample is calculated. Although the above steps can be performed manually, in a preferred method, measuring fluorescence and subtracting the fluorescence of a blank control sample from the fluorescence intensity of the reaction sample, comparing this difference to a standard and calculating the quantitative hemoglobin concentration in the test sample is done automatically with a suitably programmed microprocessor.

Thus, after cooling, the device 18 is removed from the letter

Claims (2)

7621 7 of the shell 24 (Figure 9) in which the test sample was mailed and heated and placed on a moving tray or transfer tray 26 or some other suitable means is used to transfer the sample to station 28 for fluorescence measurement. The fluorescence of the sample in each chamber 19a, 19b, 20a and 20b is determined by exposing the sample to a suitable light source system (filter or monochrometer) and a photodetector system. The measured fluorescence intensity of each chamber is then fed directly to a programmed microprocessor 29 for analysis and to calculate the quantitative concentration of hemoglobin per unit volume in the test sample. It will be appreciated that various modifications and modifications may be made to the device of the present invention without departing from the scope of the invention. Accordingly, the scope of the present invention is defined by the appended claims rather than by the description of the preferred embodiment. A sampling device for collecting a faecal test sample of a desired and predetermined size, for the quantitative determination of a test sample, the sampling device comprising an elongate sampling body (10) and a tubular part (14), characterized in that the sampling body (10) has a closed end 12) having a relatively constant diameter and shape of the outer surface throughout and having at least one grooved part (16) on the outer surface, and that the tubular part consists of a hollow sleeve (14) open at both ends, through which the entire opening extends and the shape of the inner part approximately corresponds to the diameter and shape of the sampling section, so that the