U.S. patent application number 10/079624 was filed with the patent office on 2002-08-29 for diagnostic method and apparatus.
Invention is credited to Karpas, Zeev, Lorber, Avraham.
Application Number | 20020120406 10/079624 |
Document ID | / |
Family ID | 26324005 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020120406 |
Kind Code |
A1 |
Lorber, Avraham ; et
al. |
August 29, 2002 |
Diagnostic method and apparatus
Abstract
Diagnostic method, based on the amounts of biogenic amines that
are contained in a body fluid or other sample. A number of measured
parameters related to the desired diagnostic information are
derived from the amounts. For each diagnostic information desired,
an input consisting of the identification of the diagnostic
information is provided. The input is compared to the measured
parameters and a diagnostic response is derived from the
comparison. The measured parameters may be derived from the amounts
of the biogenic amines according to a program stored in a memory.
The detection of the less volatile amines and their separation from
the more volatile ones may advantageously be enhanced by
successively adding a base and an acid, in either possible
succession, to the sample and analyzing the vapors emitted by the
sample under heating.
Inventors: |
Lorber, Avraham; (Metar,
IL) ; Karpas, Zeev; (Omer, IL) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,
KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Family ID: |
26324005 |
Appl. No.: |
10/079624 |
Filed: |
February 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10079624 |
Feb 20, 2002 |
|
|
|
09813523 |
Mar 21, 2001 |
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Current U.S.
Class: |
702/19 ;
436/111 |
Current CPC
Class: |
C12Q 1/04 20130101; Y10T
436/173845 20150115; Y10T 436/17 20150115 |
Class at
Publication: |
702/19 ;
436/111 |
International
Class: |
G06F 019/00; G01N
033/48; G01N 033/50; G01N 033/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2001 |
IL |
141233 |
Jan 31, 2002 |
IL |
PCT/IL02/00087 |
Claims
1. A method for the evaluation of a biological sample, based on the
amounts of biogenic amines, which comprises: a) determining the
amounts of said biogenic amines contained in the sample; b)
deriving from said amounts a number of measured parameters related
to the desired information; c) providing, for each information
desired, an input comprising the identification of said
information; d) comparing said input to said measured parameters;
and e) deriving from said comparison a response.
2. A method according to claim 1, wherein the evaluation is a
diagnostic evaluation, the sample is derived from the human body,
the desired information includes a diagnostic information and the
response derived is a diagnostic response.
3. A method according to claim 1, wherein the sample is a body
fluid.
4. A method according to claim 1, wherein the measured parameters
are derived from the amounts of the biogenic amines according to a
program stored in a memory.
5. A method according to claim 2, wherein the input comprising the
identification of diagnostic information is determined for a number
of expected such operations and the results are stored in a
memory.
6. A method according to claim 2, further comprising storing a
program that associates a diagnostic response to results of the
comparison of the input comprising the identification of diagnostic
information to the measured parameter, for each of the expected
diagnostic operations.
7. A method for the diagnosis of vaginal diseases which comprises
determining the presence of trimethylamine in vaginal fluid.
8. A method according to claim 7, comprising measuring the number
of ions of different amines, and if the ions of trimethylamine are
present in a number of 40% or more of the total number of all amine
ions, recognizing the presence of a vaginal disease, while if they
are present in a number of 20% or less, recognizing the absence of
a vaginal disease.
9. A method according to claim 7, further comprising measuring the
amounts of putrescine and cadaverine, and if they are abnormally
high suspecting various pathological conditions.
10. A method according to claim 1, comprising the steps of ionizing
vapors emanating from the sample and measuring the presence of
volatile amine compounds by the appearance of ions derived from
said compounds in the ion mobility measurement.
11. A method according to claim 10, further comprising enhancing
the emanation of amine vapors by the addition of reagents that
transform the amine compounds to more volatile forms.
12. A method according to claim 10, wherein the reagents are
selected from the group consisting of alkaline solutions or
ammonia.
13. A method according to claim 1, wherein the determination of the
amounts of biogenic amines comprises the steps of successively
adding a base and an acid, in either possible succession, to the
sample and analyzing the vapors emitted by the sample under
heating, to enhance the detection of the less volatile amines.
14. A method according to claim 13, wherein the vapors are also
analyzed at room temperature, to detect the more volatile
amines.
15. A method according to claim 13, wherein the less volatile
amines comprise putrescine and cadaverine.
16. A method according to claim 14, wherein the volatile amines
comprise trimethylamine.
17. A method according to claim 10, wherein the vapors are analyzed
by ion mobility spectrometry.
18. A method according to claim 13, wherein the base is an alkaline
solution and the acid is preferably a mineral acid solution.
19. A method according to claim 13, which comprises the steps of:
a--providing a sample the amines content of which is to be
determined; b--adding an acid to the sample; c--adding a base to
the sample; d--subsequently, collecting the vapors emanating from
the sample at room temperature and determining their content of
amines; e--heating the sample; and f--collecting the vapors
emanating from the samples upon heating and determining their
content of amines, wherein the order of the steps may be changed as
desired.
20. A method according to claim 10, wherein the contents of amines
are determined from the ion mobility spectra of the vapors
emanating from the sample.
21. A method according to claim 13, wherein the amount of acid used
is such as to bring the pH of the sample to a pH from about 7 to
about 1.
22. A method according to claim 19, further comprising one or more
intermediate heating steps.
23. A method according to claim 19, further comprising carrying the
gas phase emanating from the sample to a detector or a measuring
device for analyzing them.
24. A method according to claim 19, wherein the heating is carried
out so as to bring the sample to a temperature from 50.degree. C.
to 100.degree. C.
25. A method according to claim 19, wherein the heating is carried
out by immersing the sample in hot water.
26. A method according to claim 19, wherein the heating is carried
out by a means selected from the group consisting of electrical
heaters, microwave heaters, convection heaters and infrared
heaters.
27. Diagnostic apparatus, which comprises: I--an Ion Mobility
Measurement Apparatus for the determination of the amounts of
biogenic amines contained in a body fluid or other sample;
II--first elaborator means for deriving from said amounts a number
of parameters related to the desired diagnostic information;
III--buffer memory means for storing the measured parameters
derived from said determination of the amounts of biogenic amines;
IV--second elaborator means for deriving, from an input comprising
the identification of the diagnostic information desired and of the
measured parameters, a diagnostic response; and V--memory means for
storing programs controlling the operations of the first and second
elaborator means and for memorizing comparative parameters related
to said desired diagnostic information.
28. Apparatus according to claim 27, wherein the Ion Mobility
Apparatus is an Ion Mobility Spectrometer (IMS).
29. Apparatus according to claim 27, wherein the first and second
elaborator means comprises computer means.
30. Apparatus according to claim 27, wherein the first and second
elaborator means are comprised together in a single computer.
31. Apparatus according to claim 27, wherein at least one of the
first and second elaborator means consists of a LUT.
32. Apparatus according to claim 27, wherein the Ion Mobility
Spectrometer provides a spectrum of the biogenic amines and the
first elaborator means selects the amines that are relevant for the
desired diagnostic response.
33. Apparatus according to claim 32, wherein the measured
parameters are selected from the group consisting of the identity
of the ions that are present, the ratio of the numbers of different
ions that are present, the ratio of said number to the total number
of ions, and the height of the peaks of the relevant amines in the
amine spectrum.
34. Apparatus according to claim 27, wherein the memory means
contains comparative parameters for each desired diagnostic
response. 35. Apparatus according to claim 27, wherein the
comparative parameters are obtained by determining the spectra of
bodily fluids of different subjects that are free of the disease or
pathological condition for which a response is desired, determining
the measured parameters of said subjects, and averaging said
measured parameters of a sufficient number of subjects. 36.
Apparatus according to claim 35, wherein the measured parameters of
the subjects are averaged after weighting them by coefficients
which take into account the specific characteristics of each
subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of application Ser. No.
09/813,523, filed on Mar. 21, 2001.
BACKGROUND OF THE INVENTION
[0002] The invention relates to a method for diagnosing certain
pathological conditions, particularly vaginal disorders, by
determining biogenic amines in samples of body fluids or other
samples and distinguishing between more volatile and the less
volatile amine species. The invention also includes selectively
enhancing the emanation of the less volatile species and performing
a partial separation between the more volatile and the less
volatile species. The invention also relates to an apparatus for
automatically providing diagnoses of certain pathological
conditions and/or relevant diagnostic information.
[0003] It is known in the art that the presence of biogenic amines
in human body fluids may reveal or suggest pathological conditions
and dysfunctions. For example, elevated levels of certain biogenic
amines in urine may indicate the presence or the likelihood of the
presence of a cancer (there are many papers dealing with this--see,
for instance, Suh, J W, Lee, S H, Chung, B C, Park, J, Urinary
Polyamine Evaluation for Effective Diagnosis of Various Cancers,
Journal of Chromatography B, 1997, Vol. 688, Iss 2, pp. 179-186).
Several of the types of vaginal diseases may be expressed in
elevated levels of biogenic amines in vaginal discharge and fluids
(see, for instance, C. S. Chen, R. Amsel, D. A. Eschenbach and K.
K. Holmes, Biochemical diagnosis of vaginitis: determination of
diamines in vaginal fluid, J. Infectious Disease 145 (1982), pp.
337-345).
[0004] Body fluids may include e.g. urine, blood, serum, saliva,
vaginal discharge and fluids, etc.. Further, samples in which the
presence of biogenic amines may be determined may not be fluids,
but, e.g., skin and tissues, swipe samples, etc.. Even direct
sniffing of skin or breath exhaled by a subject may provide
information in this respect. This should be understood whenever
body fluids are mentioned in this application.
[0005] Chemical changes in the living system or degradation
processes of cells after death are accompanied with formation of
molecular byproducts. These processes include the breaking down of
peptides and DNA strands to smaller components, and changes in the
building blocks, amino acids, leading to the formation of amines.
Not only amino compounds are produced, but other, smaller
molecules, such as aldehydes and alcohols are also formed. One of
the processes of particular interest is the breakdown of amino
acids and the production of amines, diamines and polyamines. For
example, decarboxylation of histidine, ornithine, lysine, produces
histamine, putrescine and cadaverine, respectively.
[0006] Several analytical methods have been proposed for the
analysis of biogenic amines. Most of these are laboratory methods
that require expensive equipment, extensive sample preparation and
the skills of a trained analytical chemist or technician. Among
these are high performance liquid chromatography (HPLC), or gas
chromatography after derivatization of the samples. Biosensors may
also be used, as well as various spectrometric techniques. Solid
state sensors have also been proposed, but generally lack
specificity.
[0007] Ion Mobility Spectrometry (also, briefly, IMS) is a known
analytical method and its application for the determination of
aliphatic and aromatic amines has been suggested: see, for
instance, Z. Karpas, Ion Mobility Spectrometry of Aliphatic and
Aromatic Amines, Anal. Chem. 61 (1989), 684. An apparatus for
carrying out this method--the Ion Mobility Spectrometer (IMS)--is
used primarily for detection, identification and monitoring of
trace amounts of gases and vapors. It is particularly suitable for
detection of compounds that have high proton affinity and form
stable positive ions, or for compounds that have a high
electronegativity and readily form stable negative ions. IMS is
fully discussed in J. I. Baumbach and G. A. Eiceman, Appl.
Spectrosc. 1999, vol.53, pp.338A-355A. However, any device that may
be used for determining or measuring the mobility of ions may be
used for carrying out the invention, and therefore any reference to
IMS in this description and claims should not be construed as a
limitation, but should be construed any including instrument for
determining or measuring the mobility of ions.
[0008] The knowledge of the prior art as to the importance of
biogenic amines for the possible detection of pathological
conditions and as to the analysis of biogenic amines, including the
use of IMS, has failed so far to provide a simple and reliable
method for the diagnosis of vaginal disorders, particularly, though
not exclusively, bacterial vaginosis that affects a large number of
women. The provision of such a diagnostic method would constitute a
valuable contribution to the medical art. However, such a method is
not available: the detection of biogenic amines in vaginal fluids
is known to suggest the presence of a pathological condition, but
it does not provide specific and reliable information and merely
suggests to the specialized physician the desirability of carrying
out whatever tests and examinations may finally lead to a
diagnosis.
[0009] Samples collected from vegetation, clinical or biological
media (the samples) contain biogenic amines that arise from
degradation of amino acids through enzymatic and microbial
processes. The types of biogenic amines and their quantities are
indicative of the progress of said degradation processes, and can
thus serve as a measure for food spoilage or the existence and
extent of pathological conditions.
[0010] Some analytical methods for measuring biogenic amines, such
as gas chromatography (GC) or ion mobility spectrometry (IMS), are
based on determining their amount in the gas phase. However, while
some of these biogenic amines are highly volatile, like
trimethylamine (TMA), most have very low vapor pressures at room
temperature, and are hardly present in the gas phase. Another
reason for this is that biogenic amines may be present in the
samples as salts, such as a hydrochloride form, and not present in
the more volatile free-base structure. In such cases, their vapor
pressure may be very low, and their detection and quantification by
gas phase techniques may be very difficult.
[0011] One common practice to overcome such difficulties is to
dissociate the salts and produce the free-bases by addition of an
alkaline solution. For example, in carrying out the Amsel test for
detection of bacterial vaginosis (a common vaginal infection), the
physician adds a drop of 10% KOH solution to a swab collected from
the vaginal fluid and tries to detect the presence of volatile
amines by sniffing the sample (the so-called "whiff test").
[0012] Further, the present knowledge does not provide the
practicing physician with an apparatus for the quick diagnosis of
bacterial vaginosis and other pathological conditions, by a simple
and direct way and without the application of knowledge and
technology that are typical of different branches of science and
are not found together in any physician, no matter how competent
and dedicated. It would be extremely valuable to provide an
instrument and method whereby the average physician could obtain
from bodily fluids, quickly and in a reliable way, a diagnostic
indication of specific diseases and/or pathological conditions,
even though such a diagnostic indication may not be final and
conclusive and may require, whether positive or negative,
verification and integration.
[0013] It is therefore a purpose of this invention to provide
method for the diagnosis of vaginal disorders, particularly, though
not exclusively, bacterial vaginosis.
[0014] It is another purpose to provide a method for carrying out
such diagnosis automatically and in real time.
[0015] It is a further purpose to provide, automatically and in
real time, information of fundamental value in the diagnosis of a
variety of pathological conditions.
[0016] It is a still further purpose to overcome the problems
arising in the detection of less volatile or semi-volatile amines,
not only in clinical samples, but also in any vegetal or biological
medium.
[0017] It is a still further purpose to selectively enhance the
detection of less volatile or semi-volatile biogenic amines.
[0018] It is a still further purpose of this invention to permit to
separate, at least partially, the more volatile from the less
volatile amines.
[0019] It is a still further purpose to provide an apparatus for
the quick diagnosis of bacterial vaginosis and other pathological
conditions.
[0020] It is a still further purpose to provide a portable
instrument that is capable of carrying out the spectrometry of
bodily fluids and automatically derive from said spectrometry
significant diagnostic indications.
[0021] It is a still further purpose to provide such an instrument
that can be directed to provide diagnostic indications for specific
diseases and/or pathological conditions.
[0022] It is a still further purpose to provide such an instrument
that consists of the combination of components known in the art and
readily available.
[0023] It is a still further purpose to provide such an instrument
that can be widely used by physicians and medical institutions and
is not excessively expensive.
[0024] It is a still further purpose to provide such an instrument
which can be used, with the due precautions and warnings, by
persons other than physicians and even by the patients
themselves.
[0025] It is a still further purpose to provide such an instrument
which can be used for purposes that are not diagnostic purposes,
but are relevant to the public health, for instance, the control of
the condition of food, such as, but not exclusively, the freshness
of meat, fish and their products, as well as seafood.
[0026] It is a still further purpose to provide such an instrument
that permits instant examination of tissues removed during an
operation, or medical procedure, as an indication for malignant
tissues.
[0027] Other purposes and advantages of the invention will appear
as the description proceeds.
SUMMARY OF THE INVENTION
[0028] The invention provides a method for the diagnosis of vaginal
diseases, particularly bacterial vaginosis, which comprises
determining the presence of trimethylamine (hereinafter, TMA) ions
in vaginal fluid. According to the method, preferably, the total
amount of amine ions is measured, and if the number of TMA ions is
40% or more the total number of amine ions, the presence of
bacterial vaginosis is recognized, while if the number of TMA ions
is 20% or less, the absence of bacterial vaginosis is recognized.
The ratio of the number of ions of a given amine to the total
number of amine ions could also be called "equivalent ratio" or
"concentration of the given amine by equivalents".
[0029] Levels of putrescine and cadaverine are also measured,
according to the invention, and if the number of their ions is
above 10% of the total number of amines ions, various pathological
conditions are suspected, as will be detailed hereinafter.
[0030] A way of carrying out the method of the invention is the
following:
[0031] (1) the measurement of the presence of volatile amine
compounds, including tertiary amines like trimethylamine (TMA) and
other amines, diamines like putrescine and cadaverine as well as
polyamines like spermidine and spermine by the appearance of ions
derived from these substances in the ion mobility measurement.
[0032] (2) samples of the body (e.g. vaginal) fluid, either on a
sterile applicator or by any other method, are placed in the sample
holder or introduction system, and vapors emanating from said
sample are ionized, forming ions that are specific for the said
substances.
[0033] (3) enhancement of vapor emanation may be carried out by the
addition of an appropriate chemical reagent, that transforms the
complex amine compounds, like salts and acidic forms, to more
volatile forms. For example, this reagent can be comprised of an
alkaline solution, like KOH, NaOH and/or ammonia.
[0034] (4) a direct device for sampling vapors emanating from the
vaginal area may be used to transfer said vapors directly to the
measuring device.
[0035] (5) control of the ion chemistry by addition of a reagent
substance, comprising a volatile amine with proton affinity above
that of most common interfering compounds but below that of said
amine compounds, may be used to improve the ability to detect the
presence of said amine substances.
[0036] The invention also provides a diagnostic apparatus which
comprises:
[0037] I--an apparatus for measuring the mobility of ions, e.g. an
Ion Mobility Spectrometer (IMS) for the determination of the
amounts of biogenic amines contained in a body fluid or other
sample;
[0038] II--a first elaborator means for deriving from said amounts
a number of parameters related to the diagnostic information that
is desired in any specific case;
[0039] III--buffer memory means for storing the parameters derived
from the aforesaid determination of the amounts of biogenic
amines--hereinafter, "the measured parameters";
[0040] IV--a second elaborator means for deriving, from the input
consisting of the identification of the diagnostic information
desired and of the measured parameters, a diagnostic response.
[0041] V--memory means for storing programs controlling the
operations of the first and second eleborator and for memorizing
comparative parameters related to said diagnostic information;
[0042] The first and second elaborator means may consist of
computer means and may be comprised together in a single computer.
However, use may be made in certain cases of tables --LUTs--in
place of computers or parts of computers.
[0043] The diagnostic response may be in some cases the statement
of the presence of a disease or a pathological condition, or the
statement that such a disease or pathological condition is
suspected and its presence must be verified, or similar statements
relative to the absence of a disease or pathological condition, or
the statement that no conclusion can be drawn from the measured
parameters or that no conclusion can be drawn for other
reasons.
[0044] A similar response can be obtained from the apparatus if it
is used not for diagnostic purposes, but for checking food.
[0045] The Ion Mobility Spectrometer will provide a spectrum of the
biogenic amines, including peaks for certain amines. Ion mobility
measurements by other methods would likewise provide a quantitative
value for the presence of certain amines. The first elaborator will
be so programmed that it will firstly select the amines that are
relevant for the specific response that should be given. Based on
said selection, the elaborator will decide whether the measured
parameters should comprise the height of the peaks of the relevant
amines or areas of the spectrum about said peaks, within certain
predetermined ranges, or other parameters which will be defined by
the first elaborator program. Similar parameters, of course, may be
determined, if needed, for the amines that are not related to the
specific response to be given. Alternatively, the measured
parameters may be constituted by ratios between heights of peaks or
areas of the spectrum. The measured parameters need not be
memorized permanently, and therefore are stored in a buffer memory,
but if required, may be transferred from the buffer memory to the
permanent one.
[0046] The permanent memory of the apparatus will contain
comparative parameters for each response possibly desired.
Comparative parameters will be easily provided by determining the
spectra of bodily fluids of different subjects that are free of the
disease or pathological condition to which said response refers,
determining the measured parameters of said subjects, and averaging
said measured parameters of a sufficient number of subjects. Of
course, the average may not be a simple mathematical average, but
the measured parameters of each subject may be weighted by
coefficients which take into account the specific characteristics
of each subject, including any characteristics that are relevant to
the specific response in question. For instance, if the presence of
prostate cancer is to be determined, the age of the subjects tested
will be an extremely relevant characteristic. Of course, in such a
case, different comparative parameters may be memorized for
different ages of subjects. The physicians that will program the
first elaborator will know what characteristics are relevant and
will know how to determine the comparative parameters for each
disease or pathological condition.
[0047] The second elaborator will be programmed to determine from
the differences between the measured parameters and the comparative
parameters the likelihood of the presence of the disease or
pathological condition being considered. The program will comprise
determining the difference between said measured and said
comparative parameters, defined in any suitable way, for instance,
as a ratio of numerical values or as a value derived from a
predetermined formula relating to the specific response desired;
and deriving, from another predetermined formula or from the
response to a number of typical questions, an index of the
probability of the presence of the disease or pathological
condition in question, or a response that is more complex than the
mere indication of an index.
[0048] In its broader aspects, and beyond the specific aspect of
diagnosing vaginal diseases, particularly bacterial vaginosis and
related pathological conditions, the method of the invention
applies to any situation in which the determination of the amounts
of biogenic amines in any sample, whether derived from the human
body or having any other origin or nature, may provide valuable
information for any purpose, including but not exclusive to the
diagnosis of pathological conditions. For instance, said
information may be relevant to the use of a biological material as
food or to any other use. Therefore, in a broader aspect, the
method of the invention comprises the steps of:
[0049] a) determining the amounts of biogenic amines contained in a
biological sample;
[0050] b) deriving from said amounts a number of measured
parameters related to the desired information;
[0051] c) providing, for each information desired, an input
consisting of the identification of said information;
[0052] d) comparing said input to said measured parameters; and
[0053] e) deriving from said comparison a response.
[0054] When the method is applied to diagnostic purposes, the
sample is generally a body fluid, the desired information has a
diagnostic relevance and the response derived is a diagnostic
response. Since this is a most frequent case, reference will be
made to it hereinafter, for purposes of description and not of
limitation.
[0055] Step a) is carried out for each diagnostic operation and its
results may be stored in a buffer memory. Step b) may be carried
out according to a program stored in a buffer or permanent memory.
Step c) may be carried out distinctly for each diagnostic
operation, or may be have been carried out previously for a number
of expected such operations and the results may be stored in a
buffer or permanent memory. Step e) will be carried out according
to a stored program that will associate a diagnostic response to
results of the comparison of the aforesaid input to said measured
parameter, for each of the expected diagnostic operations; but such
a program may be derived, for particular cases, if it is not
stored. It will be obvious that, if the invention is carried out
for checking food, the diagnostic response will only consist in
classifying food according to its edibility, e.g. as safe,
doubtful, or spoiled.
[0056] A preferred embodiment of the method of the invention
comprises successively adding a base and an acid to the sample, the
amines content of which is to be determined, and analyzing the
vapors emitted by the sample under heating, so as to enhance the
detection of the less volatile or semivolatile amines (e.g.
putrescine and cadaverine). Preferably, the vapors emitted at room
temperature are analyzed as well, to enhance the detection of the
more volatile amines (e.g. TMA).
[0057] The order of the addition of base and acid is not essential:
the base may be added firstly and the acid later, or vice versa.
Preferably, the base is a strong alkaline solution, such as, e.g. a
solution of KOH or NaOH, and the acid is preferably a diluted
mineral acid, such as e.g. a 10% solution of nitric acid.
[0058] In a particular form, the method comprises the steps of:
[0059] a) providing a sample the amines content of which is to be
determined;
[0060] b) adding an acid to the sample;
[0061] c) adding a base to the sample;
[0062] d) subsequently, collecting the vapors emanating from the
sample at room temperature and determining their content of
amines;
[0063] e) heating the sample; and
[0064] f) collecting the vapors emanating from the samples upon
heating and determining their content of amines.
[0065] As hereinbefore stated, step c may precede step b, and in
general the order of the steps may be changed as desired.
[0066] If the acid is added before the base, it should be
sufficient to bring the pH, which will then generally be about 7,
down to about 1-2. Actually, for most samples, a drop of 10% nitric
acid will suffice. The amount of base added later should be
sufficient to raise the pH to about 14, in spite of the previous
addition of the acid. If the base is added first, its amount should
be sufficient to raise the pH to about 14. The subsequent addition
of acid should be in an amount that would have been sufficient, if
added before the base, to bring the pH from 7 down to 1-2. Such
subsequent addition of acid will have practically no effect on the
pH, which will remain at about 14. Therefore, no matter what the
succession of the additions of acid and base, the final pH of the
sample should be about 14. As an indication of the amount of base
involved, if 0.1 ml of a mixture of TMA, putrescine and cadaverine
is the amine component of the sample, 0.3 mL of 4N KOH could be
used. The aforesaid pH indications, however, are illustrative but
not binding, and the amounts of acid and base can be adjusted
according to cases. It can always be checked by preparing a test
sample including a known mixture of the volatile and semi-volatile
amines that are to be detected, and verifying that the amounts of
acid and base that are intended to be used will cause the emanation
of vapors, the ion mobility spectra of which are in agreement with
the known composition of said mixture.
[0067] Additional steps, particularly intermediate ones, such as a
heating, or a measurement of the vapors at room temperature,
between steps b and c, may be added to the form of the method
defined hereinbefore, for achieving additional information in
particular cases.
[0068] The determination of the contents of amines in steps d and f
is preferably carried out by determining the ion mobility spectrum
of the vapors, though it could be carried out by other, known
analysis methods, so that the reference to ion mobility spectra
should not be construed as limiting. The vapors collected at room
temperature will contain the most volatile compounds, the contents
of which will therefore be determined from said ion mobility
spectra. Heating the sample thereafter will increase the vapor
pressure of the less volatile compounds, driving them from the
sample, and causing them to enter the gas phase. The resulting
vapors will be rich in said less volatile compounds, the contents
of which will therefore be determined from the ion mobility spectra
of the vapors resulting from the heating.
[0069] Steps d and f of the process are preferably carried out by
carrying the gas phase evolved at room temperature, and then the
gas phase originating from the heating, to a detector or a
measuring device for analyzing them, preferably by ion mobility
spectrometry (IMS).
[0070] Among the volatile amines emanating from the sample at room
temperature, trimethylamine (TMA) is an important component when
the samples are body fluids collected for diagnostic purposes.
Semi-volatile amines are putrescine and cadaverine. This invention
enhances by a large factor the signal from said semi-volatile
amines and the ratio of said signal to that pertaining to TMA.
[0071] Room temperature (RT) is understood herein to be from
20.degree. C. to 30.degree. C. The heating is preferably such as to
bring the sample to a temperature from 50.degree. C. to 100.degree.
C., and is carried out by any suitable means, such as electrical
heating elements, microwave heaters, convection heaters, radiation
emitters such as e.g. infrared heaters, and so on. Another possible
way of heating the sample is to place it into a watertight
container and immerse the container in hot water (e.g.
94-95.degree. C.). When the contents of amines is determined
through the mobility spectra, the durations of stages d and f of
the treatment are such as to permit to obtain said mobility
spectra, viz. they are in the order of seconds, generally from 10
to 60 seconds.
[0072] As has been said, the analysis of the vapors obtained from
the emanation of the sample at room temperature, which may be
called "first stage vapors", is preferably carried out by IMS. A
suitable IMS apparatus is, for instance, prototype IMS (PT-IMS),
made by Rotem Industries Ltd., Israel. The same means are suitable
for analyzing the vapors obtained from the emanation of the samples
during heating, which may be called "second stage vapors".
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] The present invention can be more fully appreciated with
reference to the following detailed description, which, in turn,
refers to the drawings, in which:
[0074] FIG. 1 is a graphical representation of the biogenic amine
spectrum of the urine of a cancer patient and the comparable
spectrum of a healthy subject;
[0075] FIG. 2 is a schematic block diagram of the apparatus of the
present invention;
[0076] FIG. 3 is a graphical representation showing the peaks
indicating the evolution of amines vapors from a number of vaginal
fluid samples;
[0077] FIG. 4 is a graphical representation showing the Principal
Component (PC) analysis of a number of vaginal fluid samples
analyzed for Bacterial Vaginosis (BV);
[0078] FIG. 5 is a graphical representation showing the mobility
spectra of a sample of 0.1 mL of a mixture containing putrescine,
cadaverine, TMA (25:50:5 mmoles), obtained without the addition of
a reagent; two solid lines indicating the spectra obtained at room
temperature and under heating, while a broken line indicates the
background mobility spectrum of the instrument used;
[0079] FIGS. 6 to 13 are graphical representations of each of two
mobility spectra: one obtained at room temperature, shown in solid
lines, and one obtained during heating which raised the temperature
of the sample to about 60.degree. C., shown on broken lines; the
abscissas indicating the time in milliseconds (msec) and the
ordinates indicating the signal intensity in volts, which is
proportional to the amount of each amine emanated, under the
conditions of the various experiments;
[0080] FIG. 6 is a graphical representation showing the mobility
spectra such as those of FIG. 5, but obtained by adding KOH to the
mixture;
[0081] FIG. 7 is a graphical representation showing the mobility
spectra such as those of FIGS. 5 and 6, but obtained after adding
firstly nitric acid and then KOH;
[0082] FIG. 8 is a graphical representation showing the mobility
spectra, similar to those of FIGS. 5, 6 and 7, but of a sample of
vaginal fluid with a cotton Q-tip;
[0083] FIG. 9 is a graphical representation showing the mobility
spectra, similar to those of FIGS. 5, 6 and 7, but of another
sample of vaginal fluid with a cotton Q-tip;
[0084] FIG. 10 is a graphical representation showing the mobility
spectra, similar to those of FIGS. 5, 6 and 7, but of another
sample of vaginal fluid with a cotton Q-tip;
[0085] FIG. 11 is a graphical representation showing the mobility
spectra, similar to those of FIGS. 5, 6 and 7, but of a sample of a
piece of chicken collected after one day in a refrigerator.
[0086] FIG. 12 is a graphical representation showing the mobility
spectra, similar to those of FIGS. 5, 6 and 7, but of another
sample of a piece of chicken collected after one day in a
refrigerator.
[0087] FIG. 13 is a graphical representation showing the mobility
spectra, similar to those of FIGS. 5, 6 and 7, but of another
sample of a piece of chicken collected after one day in a
refrigerator.
DETAILED DESCRIPTION
[0088] A block diagram of an apparatus for carrying out the
invention is given in FIG. 2. In said figure, numeral 10 indicates
a chemical reaction chamber. 11 is an ion mobility measurement
device, e.g. an IMS. 12 is an analog-to-digital converter for the
acquisition, from the ion mobility measurements, of the data that
are considered relevant. 13 is a processor for processing the
acquired data according to a predetermined program. 14 is an output
device that shows the presence or absence or suspicion of presence
of predetermined pathological conditions.
[0089] The following flowsheet of operations illustrates an
embodiment of the invention:
[0090] 1) Insert sample into chemical chamber
[0091] 2) Add chemical reagent(s) to enhance emanation of volatile
compounds
[0092] 3) Transport vapors into ionization region of the IMS
[0093] 4) Ionize vapors directly and through chemical gas-phase
ion-molecule reactions
[0094] 5) Detect ions, measure their mobility and quantify
them.
[0095] 6) Acquire data or spectra
[0096] 7) Process data and compare to stored data (library, spectra
or tables).
[0097] 8) Output the result: Presence/absence of condition or
suspicion that the condition exists.
[0098] FIG. 3 comprises a diagram, showing various curves, relative
to different vaginal fluid samples. Vapors emanating from each
sample were ionized, and the amounts of ions from different amines,
particularly TMA, putrescine and cadaverine were measured. The
vapors emanate at the same time, but the ions formed from the
different compounds have different mobilities, so that when they
are measured and reported as in FIG. 3 as a function of time, the
peaks of the diagram indicate the amounts of the different amines
that are recognized from the time at which they give a signal. The
peaks relating to TMA, putrescine and cadaverine are indicated in
FIG. 3 as TMA, Di-4 and Di-5 respectively. The curves relative to
the various samples are identified by numbers at the side of FIG. 3
and it is seen that sample No. 3 has an abnormally high content of
TMA, indicating bacterial vaginosis. Curve 1 is the background
spectrum, obtained when a clean Q-tip is inserted into the chemical
reaction chamber and 300 .mu.L of 8N KOH solution are added. Curve
2 is the mobility spectrum obtained from a vaginal fluid sample of
a healthy woman with no vaginal disorder. Curve 3 was obtained from
a vaginal fluid sample of a woman diagnosed as having a vaginal
infection identified as bacterial vaginosis (BV) according to the
Amsel test. Curve 4 was obtained from a vaginal fluid sample of a
woman diagnosed as having a vaginal yeast infection. The abnormally
high level of putrescine seen in Curve 5 is indicative of an
unspecified vaginal disorder or infection.
[0099] FIG. 4 represents the scores of each mobility spectrum on
the first two principal components (PC). The first principal
component (the abscissa) represents the content of trimethylamine
(TMA) in the mobility spectrum. The second principal component (the
ordinate axis) represents the content of the diamines (putrescine
and cadaverine). The cluster of points in the upper right hand
corner (circles) is obtained from vaginal samples of women with
bacterial vaginosis. The diamond shaped data points were obtained
from vaginal samples diagnosed by the gynecologist as suffering
from some vaginal disorder (yeast, trichomonas or elevated pH
levels). The triangles represent vaginal samples taken from women
with no reported or observed vaginal disorder.
[0100] The invention can also be applied, as set forth
hereinbefore, for checking the freshness of meat.
[0101] The following examples are illustrative and in no way should
be construed as limiting the invention. A first example of the
invention will be given with reference to a diagnostic response
relative to cancer.
EXAMPLE 1
[0102] In this example, the Ion Mobility Spectrometers (IMS) used
were PhemtoChem-100 made by PCP Inc., West Palm Beach, Fla. USA and
PTIMS made by Rotem Industries, Mishor Yamin, Israel. However, any
properly equipped IMS made be used to obtain such spectra. The
first and second elaborators, in this example, are combined into a
single computer which comprises a permanent memory, a buffer
memory, a CPU, a screen, a BUS providing the necessary electrical
connections, power means, a keyboard, and all obvious accessories.
Generally, IMS may display the biogenic amine spectrum or display
their results in the form of a histogram series of bars or as a
table of compounds. In any case, the IMS transmits to the buffer
memory the amine spectrum. The operator has chosen, by means of the
keyboard, the type of response which he wants. In this case, he has
chosen a response which relates to the presence of a cancer. The
CPU is programmed to draw from the buffer memory the data of the
amine spectrum and calculate from them the measured parameters
which are relevant to the diagnosis of cancer, according to a
program which is stored in the permanent memory and which the CPU
has drawn from said memory once the operator's choice has been
made. The CPU also draws from the permanent memory the comparative
parameters and carries out the necessary comparison to draw the
response required.
[0103] FIG. 1 shows two curves relating to a healthy person and to
a cancer patient respectively, as indicated in the drawing. The
abscissa is the time at which the various amines appear and the
ordinate is their amount, and since the various amines appear at
different time, each curve constitutes an amine spectrum. The
different peaks that appear permit to diagnose the presence of a
disease, in this case cancer.
EXAMPLE 2
[0104] A sample of 0.1 mL of a mixture ("cocktail") containing
25:50:5 mmoles putrescine:cadaverine:TMA (220:510:29.5 ng
(nanograms) in sample) was analyzed by IMS.
[0105] a) FIG. 5--Without reagents at room temperature (23.degree.
C.) and during immersion in hot water (94.degree. C.). As clearly
indicated in the drawing, one curve was obtained at room
temperature and another curve was obtained when the sample was
immersed in hot water. Further, the broken line shows the
background mobility spectrum of the instrument. The biogenic amines
were not identified.
[0106] b) FIG. 6--After addition of 0.3 mL of 4N KOH solution at
room temperature (23.degree. C.) and during immersion in hot water
(94.degree. C.). The curve in solid line was obtained at room
temperature; the curve in dashed line was obtained when the sample
was immersed in hot water. A large TMA peak was observed at room
temperature, but once the sample was heated, most of the TMA was
boiled off. The other biogenic amines were not identified.
[0107] c) FIG. 7--After addition of 1 drop 10% HNO.sub.3 followed
by the addition of 0.3 mL of 4N KOH solution at room temperature
(23.degree. C.) and during immersion in hot water (94.degree. C.).
The curve in solid line was obtained at room temperature; the curve
in dashed line was obtained when the sample was immersed in hot
water. The TMA peak is similar in intensity to that in FIG. 6, but
heating results in large peaks for cadaverine and putrescine, which
could not be observed previously.
EXAMPLE 3
[0108] A 0.1 mL sample, collected by rinsing the vagina with 2 mL
of saline solution from a female patient with a vaginal infection,
was analyzed by IMS:
[0109] a) FIG. 8--Without reagents at room temperature (23.degree.
C.) and during immersion in hot water (94.degree. C.). The curve in
solid line was obtained at room temperature; the curve in dashed
line was obtained when the sample was immersed in hot water.
Biogenic amines were not identified.
[0110] b) FIG. 9--After addition of 0.3 mL of 4N KOH solution at
room temperature (23.degree. C.) and during immersion in hot water
(94.degree. C.). The curve in solid line was obtained at room
temperature; the curve in dashed line was obtained when the sample
was immersed in hot water. Biogenic amines were not identified.
[0111] c) FIG. 10--After addition of 1 drop 10% HNO.sub.3 followed
by the addition of 0.3 mL of 4N KOH solution at room temperature
(23.degree. C.) and during immersion in hot water (94.degree. C.).
The curve in solid line was obtained at room temperature; the curve
in dashed line was obtained when the sample was immersed in hot
water. The presence of putrescine was clearly observed in the
mobility spectrum of the heated sample.
EXAMPLE 4
[0112] A sample collected from a piece of chicken after one day in
a refrigerator, was analyzed by IMS:
[0113] a) FIG. 11--Without reagents at room temperature (23.degree.
C.) and during immersion in hot water (94.degree. C.). The curve in
solid line was obtained at room temperature; the curve in dashed
line was obtained when the sample was immersed in hot water. A
small TMA peak was observed, but other biogenic amines were not
identified.
[0114] b) FIG. 12--After addition of 0.3 mL of 4N KOH solution at
room temperature (23.degree. C.) and during immersion in hot water
(94.degree. C.). ). The curve in solid line was obtained at room
temperature; the curve in dashed line was obtained when the sample
was immersed in hot water. The TMA peak was larger at RT, but other
biogenic amines were not identified.
[0115] c) FIG. 13--After addition of 1 drop 10% HNO.sub.3 followed
by the addition of 0.3 mL of 4N KOH solution at room temperature
(23.degree. C.) and during immersion in hot water (94.degree. C.).
The curve in solid line was obtained at room temperature; the curve
in dashed line was obtained when the sample was immersed in hot
water. The presence of putrescine and cadaverine were clearly
observed in the mobility spectrum of the heated sample, while the
TMA peak was large at RT.
[0116] The same instrument is used for all the measurements and
therefore the background spectrum shown in FIG. 5 is not repeated
in the following figures. The instrument used was a prototype ion
mobility spectrometer (PT-IMS) made by Rotem Industries Ltd.,
Israel.
[0117] In conclusion, the signal intensities in an IMS derived from
biogenic amines without an alkali solution are negligibly small.
Addition of an alkali solution leads to enhanced emanation of
volatile amines, such as trimethylamine, while less volatile amines
are hardly seen in the mobility spectrum. Immersion in hot water
(94.degree. C.) results in boiling off the volatile compounds,
while the less volatile ones are still barely observed. However,
pretreatment with acid, followed by the alkaline solution, leads to
emanation of volatile amines at room temperature, and of the less
volatile amines when the example is immersed in hot water.
[0118] The overall effect of the process of the invention is an
enhancement of emanation of all amines by addition of an alkaline
solution (a fact well known), but also a selective enhancement of
emanation of less volatile amines by combining acid pretreatment
with heat. Thus, the presence of putrescine and cadaverine can only
readily be noticed once both pretreatment with acidification and
heat are applied to the sample.
[0119] In the drawings, the abscissa indicates time in milliseconds
and the ordinate indicates the amount of each amine emanated, under
the conditions of the various experiments, as units of volts. In
FIG. 5, a broken line indicates the background mobility spectrum of
the instrument used. The same instrument is used for all the
measurements and therefore said background spectrum is not repeated
in the following figures. The instrument used was the aforesaid
PT-IMS.
[0120] While embodiments of the invention have been described by
way of illustration, it should be understood that the invention may
be carried out with many modifications, variations and adaptations,
without exceeding the scope of the claims.
[0121] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the appended claims.
* * * * *