U.S. patent application number 11/892163 was filed with the patent office on 2008-04-17 for method for the diagnosis of helicobacter pylori infection, and a diagnostic kit for performing the method.
Invention is credited to Sitke Aygen.
Application Number | 20080090268 11/892163 |
Document ID | / |
Family ID | 27214554 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090268 |
Kind Code |
A1 |
Aygen; Sitke |
April 17, 2008 |
Method for the diagnosis of Helicobacter pylori infection, and a
diagnostic kit for performing the method
Abstract
The method for the diagnosis of Helicobacter pylori infection by
the oral administration of defined amounts of .sup.13C-labeled urea
and examination for .sup.13C content of blood samples removed at a
defined time is effected by a) removing from 0.1 to 0.6 ml of
capillary blood from the finger or ear lobe of a patient or venous
blood of a patient, in both cases with an empty stomach before the
beginning of the test; b) administering an exact amount of from 10
to 50 mg of .sup.13C-urea in aqueous solution with a pH value of 2
to 4 to the patient; c) again removing capillary or venous blood
exactly after 10 to 15 min from the administration; and d)
determining the .sup.13C content of the blood samples by isotope
ratio mass spectrometry (IRMS), and deducing the presence of
Helicobacter pylori from the increase of the .sup.13C values.
Inventors: |
Aygen; Sitke; (Cologne,
DE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
27214554 |
Appl. No.: |
11/892163 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11353126 |
Feb 14, 2006 |
|
|
|
11892163 |
Aug 20, 2007 |
|
|
|
10214323 |
Aug 8, 2002 |
7033838 |
|
|
11353126 |
Feb 14, 2006 |
|
|
|
60312541 |
Aug 16, 2001 |
|
|
|
Current U.S.
Class: |
435/29 |
Current CPC
Class: |
C12Q 1/04 20130101; G01N
33/62 20130101; G01N 2333/24 20130101; G01N 2333/205 20130101 |
Class at
Publication: |
435/029 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2001 |
DE |
101 39 299.0 |
Claims
1-5. (canceled)
6: A kit for diagnosing Helicobacter pylori infection in an adult
by blood testing comprising: a) an aqueous solution having a pH of
2 to 4 containing a dose of 10 to 50 mg of .sup.13C-urea and a
pharmacologically acceptable organic acid; b) a patient instruction
sheet for a blood test to diagnose Helicobacter pylori infection in
an adult; and c) two sample vessels for receiving blood samples,
the sample vessels being suitable for holding a strong,
non-volatile acid.
7: The diagnostic kit of claim 6 further comprising a strong,
non-volatile acid in each of the two sample vessels.
8: The diagnostic kit of claim 6, wherein the organic acid is
citric acid.
9: The diagnostic kit of claim 7, wherein the organic acid is
citric acid.
10: The diagnostic kit of claim 6 further comprising a blood
sampling device.
11: The diagnostic kit of claim 7 further comprising a blood
sampling device.
12: The diagnostic kit of claim 8 further comprising a blood
sampling device.
13: The diagnostic kit of claim 9 further comprising a blood
sampling device.
14: A kit for diagnosing Helicobacter pylori infection in an adult
by blood testing comprising: a) a 1.sup.st container holding 10 to
50 mg of .sup.13C-urea, b) a 2.sup.nd container holding a solid,
pharmacologically acceptable organic acid, c) a patient instruction
sheet for a blood test to diagnose Helicobacter pylori infection in
an adult, and d) two sample vessels for receiving blood samples,
the sample vessels being suitable for holding a strong,
non-volatile acid.
15: The diagnostic kit of claim 14, further comprising a strong,
non-volatile acid in each of the two sample vessels.
16: The diagnostic kit of claim 14, wherein the organic acid is
citric acid.
17: The diagnostic kit of claim 15, wherein the organic acid is
citric acid.
18: The diagnostic kit of claim 14, further comprising a blood
sampling device.
19: The diagnostic kit of claim 15, further comprising a blood
sampling device.
20: The diagnostic kit of claim 16, further comprising a blood
sampling device.
21: The diagnostic kit of claim 17, further comprising a blood
sampling device.
22: A kit for diagnosing Helicobacter pylori infection in an adult
by blood testing comprising: a) an aqueous solution having a pH of
2 to 4 containing a dose of 10 to less than 50 mg of .sup.13C-urea
and a pharmacologically acceptable organic acid; b) a patient
instruction sheet for a blood test to diagnose Helicobacter pylori
infection in an adult; and c) two sample vessels for receiving
blood samples, the sample vessels being suitable for holding a
strong, non-volatile acid.
23: The diagnostic kit of claim 22, further comprising d) a strong,
non-volatile acid in each of the two sample vessels; and e) a blood
sampling device.
24: A kit for diagnosing Helicobacter pylori infection in an adult
by blood testing comprising: a) a 1.sup.st container holding 10 to
less than 50 mg of .sup.13C-urea, b) a 2.sup.nd container holding a
solid, pharmacologically acceptable organic acid, d) a patient
instruction sheet for a blood test to diagnose Helicobacter pylori
infection in an adult, and e) two sample vessels for receiving
blood samples, the sample vessels being suitable for holding a
strong, non-volatile acid.
25: The diagnostic kit of claim 24, fierier comprising: f) a
strong, nonvolatile acid in each of the two sample vessels and g) a
blood sampling device.
Description
[0001] The present invention relates to a method for the diagnosis
of Helicobacter pylori infection by the oral administration of
defined amounts of .sup.13C-labeled urea and examination for
.sup.13C content of blood samples removed at a defined time.
[0002] The previously usual and, mostly performed method for the
diagnosis of Helicobacter pylori infection is the .sup.13C-urea
respiration test. This method has been described in detail in
EP-A-0 253 927. This test has the disadvantage, that it cannot be
applied for children below 3 years and adults suffering from breath
in sufficiency like asthma.
[0003] Rex Moulton-Barrett et al., The American Journal of
Gastroenterology, Vol. 88, 1993, pages 369 to 374, describe a
method in which .sup.13C-labeled hydrogencarbonate is determined in
the serum upon oral administration of .sup.13C-labeled urea. In
this test, 5 mg/kg of .sup.13C-labeled urea was administered to the
patient, and blood samples of 3 ml each were taken and examined
after 15, 30, 60, 90, 120 and 180 min. This method has been further
examined and described by Mark 3. Kim et al. in Gastroenterology
1997, 113: 31-37, W. D. Chey et al. in The American Journal of
Gastroenterology, Vol. 94, 1999, pages 1522 to 1524, Alan F.
Cuttler et al. in The American Journal of Gastroenterology, Vol.
94, 1999, pages 959 to 961, and has resulted in the introduction of
a test by the company Metabolic Solutions Inc., Nashua, N.H. In
this test, without determination of the zero value and upon
administration of a high-fat test meal "Ensure", 125 mg of
.sup.13C-urea is administered, and the .sup.13C content of 3 ml of
blood is determined after 30 min. However, due to the low accuracy
and precision and due to the very high price of the necessary
amount of .sup.13C-urea and the relatively high test quantity of
blood, this test was not successful economically, so that the
respiration test remained the mostly used test method despite of
all its disadvantages.
[0004] It has been the object of the invention to provide a method
for the diagnosis of Helicobacter pylori infection with as low as
possible an amount of .sup.13C-urea and as low as possible a blood
quantity for the examination of the .sup.13C content, which method
establishes the diagnosis of Helicobacter pylori infection more
simply, more inexpensively, more accurately and more quickly.
[0005] This object is now achieved by removing from 0.1 to 0.6 ml
of capillary blood from the finger or ear lobe of a patient or
venous blood, in both cases with an empty stomach before the
beginning of the test, administering an exact amount of from 10 to
50 mg of .sup.13C-urea in aqueous solution with a pH value of 2 to
4 to the patient, again removing capillary or venous blood exactly
after 10 to 15 min from the administration, and determining the
.sup.13C content of the blood samples by isotope ratio mass
spectrometry (IRMS), and deducing the presence of Helicobacter
pylori from the increase of the .sup.13C values.
[0006] Even when 0.6 ml of capillary blood and 50 mg of
.sup.13C-urea is used, this method is clearly superior to the
previous method, all the more so since the accuracy of the method
is enormously increased by determining the starting value, and the
method is significantly abbreviated for the patient by the removal
of a second blood sample already after 10 to 15 min. In elder
patients, the recovery of capillary blood sometimes involves
difficulties. In such cases, the method can also be performed with
the same small amount of venous blood.
[0007] Of critical importance in the method according to the
invention is the omission of the high-fat test meal. Instead, the
labeled urea is administered in an aqueous solution with a pH value
of from 2 to 4. This can be done, for example, either by adjusting
the aqueous solution of the labeled urea by means of a
non-volatile, pharmacologically acceptable organic acid to a pH
value of from 2 to 4, or by adding a pack of a solid
pharmacologically acceptable organic acid to the freshly prepared
solution. Citric acid has been found particularly suitable.
However, in principle, other organic acids of that kind, such as
ascorbic acid, are also capable of adjusting the desired pH value.
It is also possible to achieve this low pH value by the use of
orange juice, grapefruit juice or sour apple juice.
[0008] The determination of the .sup.13C content in the blood
samples can be effected, for example, by adding a strong
non-volatile acid, such as phosphoric acid, which is capable to
release the carbon dioxide in a gaseous form from the blood sample,
so that it can be measured with an IRMS device.
[0009] However, it is also possible to recover serum from the blood
samples, to remove high molecular weight components from the serum
sample by means of suitable filters, and to determine the .sup.13C
content in the remaining liquid by a preliminary elemental analysis
followed by isotope ratio mass spectrometry.
[0010] By comparative examinations, it was established that the
detection of a Helicobacter pylori infection in a respiratory test
is positive when the value of .sup.13C in the respiratory air is
about 4% above the starting values. In contrast, the method
according to the invention is capable of detecting Helicobacter
infections when the difference of the .sup.13C content is as low as
2.0%. It is obvious that the substantially lower amount of the
expensive .sup.13C-urea, on the one hand, and the quickening of the
time of the second sampling from 30 min to 10 min are of
significant importance to both the patients and the physicians. In
addition, the removal of a maximum of 0.6 ml of capillary or venous
blood is significantly simpler and more convenient than the removal
of 3 ml of venous blood in the test of the Metabolic Solutions Inc.
As compared to this test, above all, the significantly higher
accuracy and precision is of critical importance since the starting
value is determined for each patient rather than using the average
values, which are accompanied by considerable variations and
influenced by the different foods taken up by the subjects.
[0011] The diagnostic kit according to the invention for performing
the method preferably consists of an acidic aqueous solution having
a pH value of from 2 to 4 and containing exactly from 10 to 50 mg
of .sup.13C-urea, a patient instruction sheet, two sample vessels
for receiving the blood samples, and optionally a blood-sampling
device. It is decisive that the kit contains an exaxt amount of
.sup.13C-urea. A kit for children may contain less than for adults
in the over all range of 10 to 50 mg .sup.13C-urea. Also the time
difference between the two removals of blood should be exactly
measured. Different kits, however, may be run in the over all time
range of 10 to 15 min.
[0012] Another preferred embodiment comprises instead of the ready
acidic solution a container for urea with exactly from 10 to 50 mg
of .sup.13C-urea and a pack of a solid pharmacologically acceptable
organic acid, such as citric acid. Normally, 2 g of citric acid in
200 ml of water or 200 ml of orange juice, grapefruit juice, or
sour apple juice is suitable for dissolving from 30 to 50 mg of
.sup.13C-urea. Especially for children, the amount of .sup.13C-urea
can be further decreased down to 10 mg.
[0013] The blood samples preferably are taken up in commercially
available sample vessels for receiving blood samples, for example,
Vacutainer.RTM.. Preferably, they may already contain the necessary
amount of concentrated phosphoric acid, so that the CO.sub.2 is
immediately released from the blood sample. In principle, however,
it is also possible to add a strong non-volatile acid later to the
sample vessel for receiving the blood sample.
[0014] The determination of the .sup.13C content is then effected
by IRMS directly from the gas phase. An increase of the .sup.13C
content of as low as 2% after 10 min indicates infection with
Helicobacter pylori.
[0015] Alternatively, serum may also be withdrawn from the blood
sample after a short settling period. All macromolecules and
especially the lipids can be removed from this serum sample by
ultrafiltration. The remaining liquid is then first subjected to
elemental analysis by combustion, and the CO.sub.2 released thereby
is again examined by IRMS for the isotope ratio .sup.13C/.sup.12C.
In this test method, infection with Helicobacter pylori can be
detected at an increase of the .sup.13C content of as low as from 1
to 1.5%.
[0016] The higher sensitivities and accuracies of the test method
achieved according to the invention can be explained afterwards by
the fact that the .sup.13C content of the labeled urea is to a
lesser extent diluted with other carbon sources. This dilution
effect is strongest in the respiratory air test and least in the
examination of serum samples from which macromolecular molecules
are removed by ultrafiltration. Nevertheless, the method in which
CO.sub.2 is released from the blood sample by a strong non-volatile
acid and determined in the gas phase is already excellently
suitable for achieving the object of the present invention.
[0017] When the method according to the invention is performed in
practice, the blood tests are executed by the physician before and
from 10 to 15 min after administration of the .sup.13C-urea. The
Vacutainers.RTM. can be collected and sent to a central laboratory
where the determination of the .sup.13C contents is effected. From
these results fed-back to the physician, he can diagnose whether or
not there is an infection with Helicobacter pylori.
[0018] In principle, all highly sensitive isotope mass
spectrometers available on the market can be used for the IRMS
determination; however, due to their high price, they can be set up
only in central laboratories. Already for clinics, investment in
such devices cannot be expected. In contrast, the collecting and
analyzing of samples in central laboratories does not involve any
problems today in terms of logistics and prices, and the same
applies to the back transmission of analytical data to the
submitting physician or the submitting clinic.
[0019] The same is true of the determination method in the serum
from which high, molecular weight components have been removed by
ultrafiltration. The preliminary elemental analysis by combustion
can be performed in central laboratories without problems. The
recovery of serum from capillary or venous blood and
ultrafiltration can be performed without problems at least in
clinics. For the actual determination of the .sup.13C content, 20
.mu.l of serum is sufficient and will yield telling results already
for an increase of from 1 to 1.5% of .sup.13C.
* * * * *