IMPROVED METHOD FOR DETECTION OF HELICOBACTER PYLORI -GASTRITIS AND ATROPHIC GASTRITIS WITH RELATED RISKS

Abstract

According to an example aspect of the present invention, there is provided an improved method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function based on whole blood samples, sample collection time and sample storage temperature, as well as relevant biomarker content analysis.

Claims

1. A method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function, said method including the steps of: a) obtaining a whole blood sample from a subject, b) recording the time of the whole blood sample collection, c) quantitatively measuring gastrin-17 concentration of a plasma or serum sample separated from the whole blood sample, d) determining initial gastrin-17 concentration by comparing the obtained values from steps b) and c) to reference values, and e) interpreting the structure and/or function of the stomach mucosa based on said comparison.

2. A method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function, said method including the steps of: a) obtaining a whole blood sample from a subject, b) recording the storage temperature of the whole blood sample collection, c) quantitatively measuring gastrin-17 concentration of a plasma or serum sample separated from the whole blood sample, d) determining initial gastrin-17 concentration by comparing the obtained values from steps b) and c) to reference values, and e) interpreting the structure and/or function of the stomach mucosa based on said comparison

3. A method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function, said method including the steps of: a) obtaining a whole blood sample from a subject, b) recording the time of the whole blood sample collection, c) recording the storage temperature of the whole blood sample collection, d) quantitatively measuring gastrin-17 concentration of a plasma or serum sample separated from the whole blood sample, e) determining initial gastrin-17 concentration by comparing the obtained values from steps b), c) and d) to reference values, and f) interpreting the structure and/or function of the stomach mucosa based on said comparison.

4. The method according to claim 1, wherein in addition to gastrin-17 concentration also concentrations of pepsinogen I, pepsinogen II and Helicobacter pylori IgG antibodies are measured from the plasma or serum sample.

5. The method according to claim 1, wherein the whole blood sample is collected and stored without stabilizers.

6. The method according to claim 1, wherein the whole blood sample is collected and stored at temperatures between 0 to 30 C. before the separation of plasma and/or serum and analysis of the biomarker concentrations.

7. A device or a program, which determines an initial biomarker concentration based on measurement of plasma or serum biomarker concentration, time between whole blood sample collection and plasma or serum sample analysis, and storage temperature of the whole blood sample.

8. The device or a program according to claim 7, wherein the plasma or serum biomarker concentration is above the limit of quantification, the time between whole blood sample collection and plasma or serum sample analysis is between 0 to 48 hours, and the storage temperature of the whole blood sample is between 0 to 30 C.

9. The method according to claim 1 for use in a non-invasive test for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function.

Description

EMBODIMENTS

[0033] The present technology relates to the effect of temperature and time during storage and transportation of whole blood samples, when examining a person having or assuming to have the GP biomarkers indicating a Helicobacter pylori infection and/or atrophic gastritis with related risks.

[0034] It has been shown that pepsinogen I, pepsinogen II and Helicobacter pylori stands well without substantial degradation for few days storage at normal room temperature, unlike gastrin-17, as illustrated in FIG. 1. In this context different protein degradation events like oxidation, photodegradation, disulfide scrambling, deamidation, aggregation, precipitation, dissociation, fragmentation etc., are not of interest, but simply the effect of storage time and temperature.

[0035] FIG. 1 is a diagram showing relative concentration change of pepsinogens and gastrin-17, when samples are stored as whole blood at 21 C. with different period of time before analyses.

[0036] FIGS. 2 to 4 illustrate gastrin-17 concentration after storing the samples for 12, 24, 36 and 48 hours at 4 C., room temperature of 21 C., and 30 C. The relative drop of concentration is about 20%, 40% and 80% within 24 hours at 4 C., 21 C. and 30 C., respectively.

[0037] More precisely, FIG. 2a is a diagram showing the gastrin-17 concentration of samples stored as whole blood at 4 C. with different period of time before analysis

[0038] FIG. 2b is a diagram showing the gastrin-17 relative concentration change of samples as whole blood at 4 C. with different period of time before analysis.

[0039] FIG. 3a is a diagram showing the gastrin-17 concentration of samples as whole blood at 21 C. with different period of time before analysis.

[0040] FIG. 3b is a diagram showing the gastrin-17 relative concentration change of samples as whole blood at 21 C. with different period of time before analysis.

[0041] FIG. 4a is a diagram showing the gastrin-17 concentration of samples as whole blood at 30 C. with different period of time before analysis.

[0042] FIG. 4b is a diagram showing the gastrin-17 relative concentration change of samples as whole blood at 30 C. with different period of time before analysis.

[0043] Storage/transportation temperature has significant effect on the gastrin-17 degradation rate as shown in FIG. 5. Thus, FIG. 5 is a diagram showing one day (24 h) storage of whole blood in different temperatures for two different samples.

[0044] FIG. 6 is a diagram showing gastrin-17 concentration of samples stored as whole blood at 30 C. with different period of time before analyses. The dashed line presents gastrin-17 high reference of 7 pmol/l (an example of high reference; any higher concentration indicates low acid).

[0045] One embodiment of the present invention is a method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function, said method including the steps of: [0046] a) obtaining a whole blood sample from a subject, [0047] b) recording the time of the whole blood sample collection, [0048] c) quantitatively measuring gastrin-17 concentration of a plasma or serum sample separated from the whole blood sample, [0049] d) determining initial gastrin-17 concentration by comparing the obtained values from steps b) and c) to reference values, and [0050] e) interpreting the structure and/or function of the stomach mucosa based on said comparison.

[0051] Another embodiment of the present invention is a method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function, said method including the steps of: [0052] a) obtaining a whole blood sample from a subject, [0053] b) recording the storage temperature of the whole blood sample collection, [0054] c) quantitatively measuring gastrin-17 concentration of a plasma or serum sample separated from the whole blood sample, [0055] d) determining initial gastrin-17 concentration by comparing the obtained values from steps b) and c) to reference values, and [0056] e) interpreting the structure and/or function of the stomach mucosa based on said comparison

[0057] Yet another embodiment of the present invention is a method for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function, said method including the steps of: [0058] a) obtaining a whole blood sample from a subject, [0059] b) recording the time of the whole blood sample collection, [0060] c) recording the storage temperature of the whole blood sample collection, [0061] d) quantitatively measuring gastrin-17 concentration of a plasma or serum sample separated from the whole blood sample, [0062] e) determining initial gastrin-17 concentration by comparing the obtained values from steps b), c) and d) to reference values, and [0063] f) interpreting the structure and/or function of the stomach mucosa based on said comparison.

[0064] A method, wherein in addition to gastrin-17 concentration also concentrations of pepsinogen I, pepsinogen II and Helicobacter pylori IgG antibodies are determined from the plasma or serum sample, belongs to the scope of the present invention.

[0065] One suitable, but not limited to, analytical function for estimating the initial concentration of the samples is as follows. If assumed that the concentration decrease is proportional to the number of molecules

[00001]$-\frac{\mathrm{dN}}{\mathrm{dt}}N,\mathrm{or}.Math.-\frac{\mathrm{dN}}{N}=\mathrm{kdt},$

where [0066] N is the number of molecules, k indicates rate at which decrease will take place, and t is the time.
The solution to the 1.sup.st order differential equation can be presented as follows:

N(t)=N.sub.0e.sup.kt, where [0067] N.sub.0 is the initial number of molecules i.e. initial concentration of the analyte, and N(t) is the measured number if molecules (concentration).
Because the measured concentration is affected by the storage temperature, the function can be presented by general form:

N(t, T)=N.sub.0e.sup.ktf(T), where [0068] T is the storage temperature of the sample.
The concentration half time t.sub.1/2 can be solved from the formula:

[00002]$k\left(T\right)=\frac{t_{\frac{1}{2}}}{\ln \left(2\right)}.Math.f\left(T\right)$

The half time

[00003]$t_{\frac{1}{2}}$

and k(T) can be estimated from the measured data.

[0069] As the concentration drops monotonically vs time and temperature, and if the concentration is higher than the Limit of Quantification (LoQ), and information of the storage temperature and time of sample collection is known, it is possible to estimate the initial concentration and use that information when interpreting the results.

[0070] According to one embodiment of the present invention, high (vs the reference range) gastrin-17 concentration indicates acid-free stomach. If a sample that has been stored for some time before measurement still gives high concentration value, also the initial concentration must have been high. Such a situation is illustrated by the sample BN1999 in the FIG. 5, assuming that measurement have been carried out next day (24 hours) from sample collection. The interpretation on stored sample results is as valid as if the sample would have been analyzed right after collection.

[0071] Further, if it is sure that the temperature chain has not exceeded certain temperature, it can predict concentration vs. reference range, and enable the use of that when interpreting the results. For example, when sample BN1998 in FIG. 5 has been stored at room temperature (21 C.) before measurement, and the measured value is 5 pmol/l, it can be assumed that the half time is less than 24 hours, and therefore be confident that the initial value has been higher than the example high reference of 7 pmol/l. Thus, also this case indicates lack of acid in the stomach and e.g. with low pepsinogen level will increase the specificity of atrophic gastritis diagnosis.

[0072] Furthermore, if it is assumed that the sample has been stored for 24 hours before measurement, and the value is about 2 pmol/l, it indicates that the acid content of the stomach is normal, less than the upper limit of the reference range.

[0073] However, if the measured concentration is below the LoQ, it is not possible to predict the initial concentration. In that case the gastrin-17 results cannot be used for the interpretation. The GastroPanel interpretation must then be made based on the other biomarker values.

[0074] According to one embodiment the whole blood sample is collected and stored without stabilizers.

[0075] According to one embodiment the time period between whole blood sample collection and further analysis from the plasma or serum sample is between 0 to 120 hours, more preferably between 0 to 72 hours and most suitably between 0 to 48 hours.

[0076] According to one embodiment the whole blood sample is collected and stored at temperatures between 0 to 30 C., for example at 4 C., 21 C. or 30 C., before the separation of plasma and/or serum and analysis of the biomarker concentrations.

[0077] A device or a program which determines an initial biomarker concentration based on measurement of plasma or serum biomarker concentration, time between whole blood sample collection and plasma and/or serum sample analysis, and storage temperature of the whole blood sample belongs to the scope of the present invention.

[0078] Preferably, the plasma or serum biomarker concentration is above the limit of quantification, the time between whole blood sample collection and plasma and/or sample analysis is between 0 to 48 hours, and the storage temperature of the whole blood sample is between 0 to 30 C.

[0079] Thus, such device or program may be for example GastroSoft program, which is designed for interpreting GastroPanel results, which has been modified to take into consideration the above mentioned essential parameters of the present invention.

[0080] One further embodiment of the present invention is to use the methods as disclosed in a non-invasive test for screening of asymptomatic and symptomatic subjects with Helicobacter pylori infection, atrophic gastritis with related risks and/or disturbed gastric function.

[0081] It is to be understood that the embodiments of the invention disclosed are not limited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0082] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases in one embodiment or in an embodiment in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed.

[0083] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0084] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0085] While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0086] The verbs to comprise and to include are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of a or an, that is, a singular form, throughout this document does not exclude a plurality.

INDUSTRIAL APPLICABILITY

[0087] At least some embodiments of the present invention find industrial application for example in screening and examination of patients with dyspepsia, as well as for the screening and detection of atrophic gastritis (AG) with related risks, such as stomach- and oesophageal cancer.

CITATION LIST

Patent Literature

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