DIAGNOSTIC COMPOSITION FOR IDENTIFYING AMNIOTIC FLUID
FIELD OF THE INVENTION
The present invention relates to the field of medical diagnostics and more
specifically, to identification of amniotic fluid.
BACKGROUND OF THE INVENTION
Premature rupture of the amniotic sac is a common disorder that happens to occur
in around 10% of pregnant women. Failure to treat premature rapture may cause intraamnion
infection, disruption in fetal lung development and even perinatal death.
Several approaches for detecting amniotic leakage are known in the art, including
chromatographic detection of a color change in a diagnostic article following contact with
vaginal secretion.
A number of devices involving panty shields, swabs and the like, with indicators
aimed to identify amniotic fluid were developed, for example, in U.S. Patent Nos.
5,897,834; 6,126,597 and 6,149,590. These devices can be worn, or otherwise used, by the
user and whenever there is a secretion it is absorbed and further detected.
US Patent No. 6,921,647 and Continuation-in-Parts thereof, including, US Patent
Nos. 7,314,752; 7,541,177 and 7,947,467 disclose secretion-monitoring article for
identifying a secreted biological fluid, including, amniotic fluid, comprising: a body that
includes an absorbent material and an indicator system having at least one pH determining
member. The pH determining member includes a chemical composition which reacts with
fluids containing urea differently than with fluids that do not contain urea, in a manner
such that the former can be distinguished from the latter while avoiding false positive
indications. The pH determining member may further, or alternatively, include a
hydrophobic chemical composition that reacts with fluids based on the buffer capacity
thereof, such that fluids having different buffer capacities can be distinguished.
It has been also proposed to detect the rupture of the amniotic sac by employing
immunochemical analysis of the proteins in the amniotic fluid, such as, alpha-fetoprotein,
prolactin, fibronectin, and insulin-like growth-factor binding protein 1. For example, US
Patent No. 8,114,610 discloses a method of detecting amniotic fluid in a sample
comprising a vaginal secretion of a pregnant woman using a combination of PAMG-1-
specific monoclonal antibodies.
There is an unmet need for an improved diagnostic system that can differentiate
between amniotic fluid and an interfering biological fluid, such as, urine, with minimal
false positive results, while providing a reliable indication which is stable over time, and
while reducing the amount of time required to get the reliable result.
SUMMARY OF THE INVENTION
The present invention discloses an indicator composition for detecting amniotic
fluid in vaginal secretion comprising: a plurality of polymers; a plasticizer; a surfactant; an
ion-balance reagent; an acid and an indicator agent, wherein said composition is devoid of
a wetting agent. The present invention further discloses diagnostic articles for identifying
amniotic fluid and methods of using same.
Advantageously, the articles of the invention may operate right after contacting the
vaginal secretion. Specifically, the articles and diagnostic compositions disclosed herein
do not require any drying time and/or do not require waiting for urine evaporation. Thus,
detection of amniotic fluid (or other fluids) by the articles and compositions disclosed
herein is a one-step process, namely, upon contact of liquid (amniotic fluid or urine) with
an article comprising the diagnostic composition, detection can be carried out on the
article as is, right after being in contact with the fluids. Without wishing to be bound by
any theory or mechanism, it is assumed that this advantage is rendered by the unique
combination of the ingredients in the diagnostic formulations of the invention, or may be
specifically attributed to the lack of wetting agent, the presence of an aromatic carboxylic
acid, the presence of an hydrophilic non-ionic polymer as surfactant, the presence of a triester
non-cyclic plasticizer or the combination thereof, together with the rest of the
ingredients.
Furthermore, the articles of the invention enable initial self-detection and do not
require the patient to visit health-care professional in order to distinguish between positive
and false-positives. Accordingly, using the article of the invention saves a significant
amount of time required to determine if amniotic fluid is actually leaking.
In some embodiments, there is provided an indicator composition for detecting
amniotic fluid in vaginal secretion comprising: a plurality of polymers; a non-cyclic
plasticizer; a hydrophilic surfactant; an aromatic carboxylic acid; an ion-balance reagent
and an indicator agent, wherein said composition is devoid of a wetting agent.
In some embodiments, said aromatic carboxylic acid comprises salicylic acid,
benzoic acid or a combination thereof.
In some embodiments, the aromatic carboxylic acid is salicylic acid.
In some embodiments, the hydrophilic surfactant is a non-ionic surfactant.
In some embodiments, the hydrophilic surfactant is a polymer.
In some embodiments, the hydrophilic surfactant comprises a block copolymer
surfactant.
In some embodiments, the indicator agent is negatively charged.
In some embodiments, the ion-balance reagent is a quaternary ammonium salt.
In some embodiments, the non-cyclic plasticizer is a tri-ester plasticizer.
In some embodiments, there is provided an indicator composition for detecting
amniotic fluid in vaginal secretion consisting of a plurality of polymers; a non-cyclic
plasticizer; a hydrophilic surfactant; an aromatic carboxylic acid; an ion-balance reagent
and an indicator agent.
In some embodiments, there is provided an article for detecting amniotic fluid in
vaginal secretion comprising: an absorbent material for absorbing a biological fluid
secreted from a person, and an indicator composition, comprising: a plurality of polymers;
a non-cyclic plasticizer; an hydrophilic surfactant; an aromatic carboxylic acid; an ionbalance
reagent and an indicator agent, wherein said composition is devoid of a wetting
agent.
In some embodiments, the absorbent material is selected from the group consisting
of a swab, gauze, panty shield, hygienic napkin, diaper and interlabial absorbent structure.
In some embodiments, there is provided a method for diagnosing amniotic fluid
leakage in vaginal secretion comprising:
(a) positioning a secretion-monitoring article to receive a biological
fluid secreted from the subject, the article comprising an indicator composition
comprising: a plurality of polymers; a non-cyclic plasticizer; an hydrophilic
surfactant; an aromatic carboxylic acid; an ion-balance reagent and an indicator
agent, wherein said composition is devoid of a wetting agent;
(b) contacting said article with vaginal secretion; and
(c) viewing the article, wherein a color change indicates the presence of
amniotic fluid.
In some embodiments, said color change is a dark stain relative to background of
the secretion monitoring article. In some embodiments, the dark stain is a dark blue stain
or a dark green stain.
In some embodiments, said viewing is performed within less than 10 minutes after
said contacting. In some embodiments, said viewing is performed within less than 9
minutes after said contacting. In some embodiments, said viewing is performed within
less than 8 minutes after said contacting. In some embodiments, said viewing is performed
within less than 7 minutes after said contacting. In some embodiments, said viewing is
performed within less than 6 minutes after said contacting. In some embodiments, said
viewing is performed within less than 5 minutes after said contacting. In some
embodiments, said viewing is performed within less than 4 minutes after said contacting.
In some embodiments, said viewing is performed within less than 3 minutes after said
contacting. In some embodiments, said viewing is performed within less than 2 minutes
after said contacting.
In some embodiments, said indicator composition is consisting of a plurality of
polymers; a non-cyclic plasticizer; a hydrophilic surfactant; an aromatic carboxylic acid;
an ion-balance reagent and an indicator agent.
In some embodiments, there is provided a kit comprising the article for detecting
amniotic fluid in vaginal secretion, and instructions for use.
In some embodiments, the kit further comprises a color scale presenting the color
expected to appear upon contact of the article with amniotic fluid and the color expected
to appear upon contact of the article with urine and any non-amniotic fluid.
Further embodiments, features, advantages and the full scope of applicability of the
present invention will become apparent from the detailed description and drawings given
herein after. However, it should be understood that the detailed description, while
indicating preferred embodiments of the invention, are given by way of illustration only,
since various changes and modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
Exemplary embodiments are illustrated in referenced figures. It is intended that the
embodiments and figures disclosed herein are to be considered illustrative rather than
restrictive. The figures are listed below.
Fig. 1 shows the relative specificity of the articles disclosed herein (including
ALM1 which is also termed "AL-SENSE Blue") at the following time points: 0-2 min., 5
min., 10 min., 20 min., 30 min 1 hr. and 2 hrs.
Fig. 2 exhibits the results obtained from contacting urine samples (pH 7) with one
articles encompassing candidate composition - ALM1 and test compositions 3Marl and
3Mar2.
Fig. 3A demonstrates a photo of an absorbent article containing the ALM1
formulation 30 min. following contact with amniotic fluid.
Fig. 3B demonstrates a photo of an absorbent article containing the ALM1
formulation 30 min. following contact with urine.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an indicator composition, a diagnostic article
comprising same, and methods of use thereof for obtaining an immediate, clear and
reliable identification of amniotic fluid leakage.
The terms "amniotic fluid leakage" and "premature rupture of the amniotic sac" are
interchangeable and relate to the spontaneous rupture of the membranes (the 'amniotic sac'
is often termed 'fetal membrane') 24 or more hours before the onset of labor. About 30-
leakage is associated with a significant increase in the risk of an intrauterine infection and
disturbance of development of the fetal lung system, definitive and reliable diagnosis of
the rupture is material. Intrauterine penetration of such infections increases both maternal
and perinatal morbidity and mortality by about ten percent. Immediate diagnosis of a
rupture at 38 to 40 weeks of pregnancy is crucial, since once detected delivery should be
induced as soon as possible. The rupture diagnosis is also important before 37 weeks of
pregnancy because it enables prevention of intra-amnion infection and the stimulation of
fetal lung development.
Due to the severe consequences of amniotic fluid leakage, pregnant women
undergo severe stress and often go to a health-care professional upon secretion of any
liquid from the vicinity of the vagina. The health-care professional looks for the presence
of amniotic fluid by checking the pH of the vaginal secretions - amniotic fluid is having a
pH of between 6.7 and 7.5. Since pregnant women often have urinary incontinence and
since urine typically has a pH of between 5.0 and 8.0, if only pH is checked, a false
positive result may occur: urine being identified as amniotic fluid. Consequently, it is
necessary that a vaginal secretion be examined using a microscope for the presence of a
fern-shaped pattern indicative of amniotic fluid.
As the time between the fluid secretion and the arrival at the health-care
professional may be long, there is often no evidence of amniotic fluid upon examination.
The secretion may mistakenly assumed to be urine, which may result with tragic
consequences. On the other hand, the healthcare professional may decide to err on the side
of caution, misdiagnosing the secretion of urine as amniotic fluid leading to an
unnecessary hospitalization and patient stress.
Various diagnostic indicators are known, however, an obstacle of many is that they
often provide "false positives" due to changes in pH on drying, interfering biological
fluids and repetitive cycles of drying/wetting. The "false positive" readings can be
stressful and time consuming to the user. A device that minimizes these "false positive"
readings is disclosed herein. The present invention is an improvement over the prior art,
providing a diagnostic composition which is more accurate, reliable and convenient for the
user. The diagnostic composition of the present invention comprises an indicator agent
that identifies the pH associated with the fluid, a plurality of polymers, a tri-ester noncyclic
plasticizer, hydrophilic non-ionic polymer surfactant, an ion-balance reagent and at
least one aromatic carboxylic acid, wherein said composition is devoid of a wetting agent.
As used herein and in the claims the term "plurality" means at least two.
In some embodiments, there is provided an indicator composition for detecting
amniotic fluid in vaginal secretion comprising: a plurality of polymers; a non-cyclic
plasticizer; a hydrophilic surfactant; an aromatic carboxylic acid; an ion-balance reagent
and an indicator agent, wherein said composition is devoid of a wetting agent.
In some embodiments there is provided an indicator composition for detecting
amniotic fluid in vaginal secretion comprising: a plurality of polymers; a tri-ester noncyclic
plasticizer; a hydrophilic non-ionic polymer surfactant; an aromatic carboxylic
acid; an ion-balance reagent and an indicator agent, wherein said composition is devoid of
a wetting agent.
In some embodiments, the indicator composition is consisting of a plurality of
polymers; a non-cyclic plasticizer; a hydrophilic surfactant; an aromatic carboxylic acid;
an ion-balance reagent and an indicator agent.
In some embodiments, the indicator composition is consisting of a plurality of
polymers; a tri-ester non-cyclic plasticizer; a hydrophilic non-ionic polymer surfactant; an
aromatic carboxylic acid; an ion-balance reagent and an indicator agent.
In some embodiments there is provided an article for detecting amniotic fluid in
vaginal secretion comprising: an absorbent material for absorbing a biological fluid
secreted from a person, and an indicator composition comprising a plurality of polymers; a
non-cyclic plasticizer; a hydrophobic surfactant; an aromatic acid; an ion-balance reagent
and an indicator agent, wherein said composition is devoid of a wetting agent.
In some embodiments there is provided a method for diagnosing amniotic fluid
leakage in vaginal secretion comprising:
(a) positioning a secretion-monitoring article to receive a biological fluid secreted
from the subject, the article comprising: an absorbent material and an indicator
composition comprising a plurality of polymers, an aromatic acid, a non-cyclic
plasticizer, a hydrophobic surfactant, an ion-balance reagent and an indicator
agent, wherein said composition is devoid of a wetting agent;
(b) contacting said article with vaginal secretion; and
(c) viewing the article, wherein a color change indicates the presence of amniotic
fluid.
In some embodiments, contacting the article comprises removing release tapes
covering adhesive strips, thereby exposing the adhesive strip, and placing the article in the
crotch portion of an undergarment, thereby attaching said article to said crotch portion of
the undergarment.
In some embodiments, viewing the color change comprises detecting dark staining
on the article, wherein the dark staining is indicative of the presence of amniotic fluid.
In some embodiments, the dark staining is blue staining. In some embodiments, the
dark staining is green staining.
The compositions described herein may be prepared by methods well known to
those skilled in the art.
In some embodiments, said plurality of polymers comprises a first polymer and a
second polymer, the hydrophobicity of said first polymer is higher than the hydrophobicity
of said second polymer.
In some embodiments, said plurality of polymers comprises a first polymer and a
second polymer having similar hydrophobicity. The terms "hydrophobic" and
"hydrophilic" polymers refer on the amount of water vapor absorbed by the polymers at
100% relative humidity. Thus, "hydrophobic polymers" typically refer to polymers that
absorb only up to 1 wt. % water at 100% relative humidity, while moderately hydrophilic
polymers absorb 1-10% wt. % water and hydrophilic polymers are capable of absorbing
more than 10 wt. % of water. In some embodiments, the polymers of the invention are
water-insoluble polymers. In some embodiments, each of the polymers comprises
cellulose, derivatives thereof or copolymers thereof, including, without limitation,
cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellophane,
cellulose nitrate, cellulose propionate, cellulose propionate butyrate, cellulose diacetate,
cellulose triacetate, cellulose ethers, and carboxymethyl cellulose, and any copolymers and
any blends of the aforementioned polymers.
In some embodiments, each polymer comprises cellulose, derivatives thereof or
copolymers thereof.
In some embodiments, the polymers of the invention are cellulose esters
comprising partially acetylated cellulose polymeric chains, including, but not limited to,
cellulose acetate in which substantially each one of positions 2, 3 and 6 of the monomeric
glucose units of the cellulose polymer, consists of either free hydroxyl or acetate. In some
embodiments, the polymers are cellulose esters comprising substantially acetylated
cellulose polymeric chains, including, but not limited to, cellulose triacetate. In some
embodiments, the polymers are cellulose esters comprising partially esterified cellulose
polymeric chains, wherein the esterification comprises partial acetylation and partial
butyrilation, including, but not limited to, cellulose acetate butyrate, in which substantially
each one of positions 2, 3 and 6 of the monomeric glucose units of the cellulose polymer
consists of either free hydroxyl, acetate or butyrate. In some embodiments, the polymers
are cellulose esters comprising partially esterified cellulose polymeric chains, wherein the
esterification comprises partial acetylation and partial propionilation, including, but not
limited to, cellulose acetate propionate, in which substantially each one of positions 2, 3
and 6 of the monomeric glucose units of the cellulose polymer consists of either free
hydroxyl, acetate or propionate.
Without wishing to be bound by any theory or mechanism of action, the less
hydrophobic polymer, is more hydrophilic thereby improves the penetration of the fluid to
be tested, into the panty liner. In addition, the more hydrophobic polymer provides
improved adhesion of the indicator composition to the absorbent carrier (e.g. the fabric)
thereby minimizing or even avoiding leaching of the formulation components.
In some embodiments, said plurality of polymers comprises at least one polymer
selected from the group of cellulose acetate and cellulose acetate butyrate.
In some embodiments, said plurality of polymers comprises a first polymer, such
as, cellulose acetate butyrate, and a second polymer, such as, cellulose acetate, the
hydrophobicity of said first polymer is higher than the hydrophobicity of said second
polymer.
In some embodiments, the plasticizer is a low molecular weight plasticizer.
Typically, a low molecular weight plasticizer is a plasticizer having a molecular weight of
less than 600 gr/mol. In some embodiments, the plasticizer is having a molecular weight
of less than 500 gr/mol. In some embodiments, the plasticizer is having a molecular
weight of less than 400 gr/mol. In some embodiments, the plasticizer is having a
molecular weight of less than 300 gr/mol.
Suitable low molecular weight plasticizers include alkyl and aryl phosphates, such
as, tributyl phosphate, trioctyl phosphate, tricresyl phosphate, and triphenyl phosphate;
alkyl citrate and citrate esters, such as, trimethyl citrate, triethyl citrate, tributyl citrate,
acetyl triethyl citrate, and trihexyl citrate; dialkyladipates such as dioctyladipate (DOA;
also referred to as bis(2-ethylhexyl)adipate), diethyl adipate, di(2-methylethyl)adipate, and
dihexyladipate; dialkyl tartrates such as diethyl tartrate and dibutyl tartrate;
dialkylsebacates such as diethyl sebacate, dipropylsebacate and dinonylsebacate; dialkyl
succinates such as diethyl succinate and dibutyl succinate; alkyl glycolates, alkyl
glycerolates, glycol esters and glycerol esters such as glycerol diacetate, glycerol triacetate
(triacetin), glycerol monolactatediacetate, methyl phthalyl ethyl glycolate, butyl phthalyl
butyl glycolate, ethylene glycol diacetate, ethylene glycol dibutyrate, triethylene glycol
diacetate, triethylene glycol dibutyrate and triethylene glycol dipropionate; acetylated
monoglycerides and mixtures thereof.
In some embodiments, the plasticizers comprise low molecular weight plasticizers,
including, but not limited to, alkyl citrate and citrate esters, such as, trimethyl citrate,
triethyl citrate, tributyl citrate, acetyl triethyl citrate, and trihexyl citrate, acetylated
monoglycerides and mixtures thereof.
In some embodiments, the non-cyclic plasticizer is a tri-ester plasticizer. In some
embodiments, the low molecular weight plasticizers comprise a triester structure. Such
plasticizers include, but are not limited to, alkyl citrate and citrate esters, such as, trimethyl
citrate, triethyl citrate, tributyl citrate, acetyl triethyl citrate and trihexyl citrate. In some
embodiments, the plasticizer is triethyl citrate. In some embodiments, the plasticizer is
other than dibutyl phthalate. In some embodiments, the plasticizer is other than Hexamoll
Dinch (1,2-cyclohexane dicarboxylic acid diisononyl ester).
In some embodiments, the non-cyclic plasticizer comprises triethyl citrate. In some
embodiments, the amount of the surfactant is within the range of 1% to 10% (wt/wt),
relative to the total weight of the solid components.
The term "solid components" or "total solids" as used herein refer to the all
ingredients within each formulation, in the absence of solvent, in particular, water and/or
acetone.
The term "surfactant" as used herein refers to one or more surfactants. In some
embodiments, surfactants refer to a plurality of surfactants.
In some embodiments, the surfactant comprises a hydrophilic surfactant. In some
embodiments, the hydrophilic surfactant is a polymer. In some embodiments, the
surfactant comprises a block copolymer surfactant. In some embodiments, the polymeric
surfactant comprises a tri-block copolymer surfactant. In some embodiments, the
polymeric surfactant comprises poly(ethylene oxide) and/or copolymers thereof. In some
embodiments, the polymeric surfactant comprises poly(propylene oxide) and/or
copolymers thereof. In some embodiments, the block copolymer surfactant comprises
copolymer of poly(ethylene oxide) and poly(propylene oxide) and/or copolymers thereof.
In some embodiments, the hydrophilic surfactant is a non-ionic surfactant. In some
embodiments, the non-ionic surfactant comprise a non-ionic polymeric surfactant. In some
embodiments, the surfactant is selected from the group of poloxamer, alkyl poly(ethylene
oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide), octylglucoside,
decylmaltoside, cetyl alcohol, oleyl alcohol, cocamidemonoethanolamine (cocamide
MEA), cocamidediethanolamme (cocamide DEA), cocamidetriethanolamme (cocamide
TEA), polysorbate, such as, Tween® (e.g. Tween 20® or Tween 80®), lecithin and
combinations thereof.
In some embodiments, the non-ionic polymeric surfactant is selected from the
group of poly(ethylene oxide) and copolymers of poly(ethylene oxide), poly(propylene
oxide), polysorbate, such as, Tween® (e.g. Tween 20® or Tween 80®) and combinations
thereof.
In some embodiments, the surfactant comprises poloxamer, polysorbate and a
combination thereof.
In some embodiments, the surfactant comprises a poloxamer. In some
embodiments, the surfactant comprises poloxamer 407.
In some embodiments, the surfactant comprises a polysorbate. In some
embodiments, the non-ionic surfactant is other than ionic surfactants, such as, aliquot 336.
The term "poloxamer" as used herein refers to nonionic triblock copolymers
comprising a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) and
two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are
commonly termed "P" followed by three digits, the first two times 100 refer to the
estimated molecular mass of the polyoxypropylene core, and the last digit times 10 is the
percentage polyoxyethylene content. For example, P407 refers to a poloxamer with a
polyoxypropylene molecular mass of 4,000 g/mol and 70% polyoxyethylene content.
The term "polysorbate" as used herein generally refers to polyoxyethylene
derivative of sorbitan monolaurate. Common commercial brand names for polysorbates
include Tween®, such as, Tween 20® or Tween 80®. The number following the
polyoxyethylene part refers to the total number of oxyethylene groups found in the
molecule. The number following the polysorbate part is related to the type of fatty acid
associated with the polyoxyethylene sorbitan part of the molecule. Monolaurate is
indicated by 20, monopalmitate is indicated by 40, monostearate by 60 and monooleate by
80.
Without wishing to be bound by any theory or mechanism, the non-ionic polymeric
surfactant distributes within the urine and as a result retention time of urine is reduced
thereby avoiding false positive results from urine.
In some embodiments, the at least one carboxylic acid comprises at least one
unsubstituted or substituted carbocyclic or heterocyclic carboxylic acid. In some
embodiments, the at least one carboxylic acid comprises at least one of unsubstituted or
substituted aromatic or heteroaromatic carboxylic acid. In some embodiments, the at least
one carboxylic acid comprises at least one unsubstituted or substituted aromatic
carboxylic acid. In some embodiments, the at least one substituted carboxylic acid is
selected from the group consisting of salicylic acid, benzoic acid, phthalic acid,
terphthalic acid, isophthalic acid, a-naphthoic acid, -naphthoic acid, isomers of toluic
acid, isomers of halobenzoic acid, such as, but not limited to /?-fluorobenozic acid and mchlorobenzoic
acid, isomers of nitrobenzoic acids, isomers of trifluoromethylbenzoic acid,
isomers of aminobenzoic acid, such as, but not limited to, anthranilic acid, isomers of
anisic acid, m-hydroxybenzoic acid and /?-hydroxybenzoic acid. Each possibility
represents as separate embodiment of the present invention.
The term "isomer" as used herein refers to a positional isomer, denoting a relative
location in which two or more substituents are located on the aromatic ring.
In some embodiments, the acid is an aromatic carboxylic acid. In some
embodiments, the aromatic carboxylic acid is in the form of Ar-COOH. In some
embodiments, Ar is selected from the group consisting of aryl and heteroaryl rings,
wherein Ar is unsubstituted or substituted. Non limiting examples of substitutions include
halogen, haloalkyl, C1-C4 alkyl, aryl, heterocyclyl, hydroxy, alkoxy, aryloxy, alkylaryloxy,
heteroaryloxy, oxo, cycloalkyl, phenyl, heteroaryl, naphthyl, amino, alkylamino,
arylamino, heteroarylamino, dialkylamino, diarylamino, alkylarylamino,
alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxy, carbamoyl,
carboxamide, cyano, sulfonyl, sulfonylamino, sulfinyl, sulfinylamino, thiol, Ci to o
alkylthio, arylthio, or d to d o alkylsulfonyl groups. Any substituent can be unsubstituted
or further substituted with any one of these aforementioned substituents. Each possibility
represents as separate embodiment of the present invention.
The terms "aryl" and "Ar" as used herein alone or as part of another group are
interchangeable and denote an aromatic ring system containing from 6-14 ring carbon
atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic and the like. Non-limiting
examples of aryl groups are phenyl, naphthyl, including, 1-naphthyl and 2-naphthyl, and
the like. The aryl group may be unsubstituted or substituted through available carbon
atoms with one or more groups defined hereinabove for alkyl.
The term "heteroaryl" used herein alone or as part of another group denotes a
heteroaromatic system containing at least one heteroatom ring atom selected from
nitrogen, sulfur and oxygen. The heteroaryl generally contains 5 or more ring atoms. The
heteroaryl group can be monocyclic, bicyclic, tricyclic and the like. Also included in this
expression are the benzoheterocyclic rings and other fused heterocyclic ring systems. If
nitrogen is a ring atom, the present invention also contemplates the N-oxides of the
nitrogen containing heteroaryls. Non-limiting examples of heteroaryls include thienyl,
benzothienyl, 1-naphthothienyl, thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl,
pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl,
purinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,
pteridinyl, carbolinyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl and the like. The
heteroaryl group may be substituted through available atoms with one or more groups
defined hereinabove for alkyl.
The terms "heterocyclic ring" or "heterocyclyl" used herein alone or as part of
another group denote a five-membered to eight-membered rings that have 1 to 4
heteroatoms, such as oxygen, sulfur and/or nitrogen, in particular nitrogen, either alone or
in conjunction with sulfur or oxygen ring atoms. These five-membered to eightmembered
rings can be saturated, fully unsaturated or partially unsaturated. Preferred
heterocyclic rings include piperidinyl, pyrrolidinyl, pyrrolinyl, pyrazolinyl, pyrazolidinyl,
piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl,
dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl,
dihydropyranyl, tetrahydropyranyl, and the like. The heterocyclyl group can be
unsubstituted or substituted through available atoms with one or more groups defined
hereinabove for alkyl.
The term "halogen" used herein alone or as part of another group includes chloro,
fluoro, bromo, and iodo.
In some embodiments, the at least one carboxylic acid comprises at least one
carboxylic acid having a pKa smaller than 3. In some embodiments, the at least one
carboxylic acid comprises at least one carboxylic acid having a water solubility of less
than 100 gr/L. In some embodiments, the at least one carboxylic acid comprises at least
one carboxylic acid having a water solubility of less than 10 gr/L. In some embodiments,
the at least one carboxylic acid comprises at least one carboxylic acid having a water
solubility of less than 5 gr/L.
In some embodiments, the aromatic carboxylic acid is selected from the group
consisting of salicylic acid, benzoic acid and the like and combinations thereof. Each
possibility represents as separate embodiment of the present invention.
In some embodiments, the aromatic carboxylic acid is salicylic acid.
In some embodiments, the aromatic carboxylic acid is other than non-aromatic
carboxylic acids, such as, lactic acid, oxalic acid or citric acid. In some embodiments, the
at least one carboxylic acid is other than citric acid.
Without being bound by any theory or mechanism, the aromatic carboxylic acid
creates a complex, and may function as a trap, with the ammonium ions within the urine.
This interaction results in reduction of interference from urine, including urine with pH
level of 7.0 units, and even higher, and therefore improves indicator specificity.
In some embodiments, the ion-balance reagent is in an amount of about 0.1% to
2%. In some embodiments, the ion-balance reagent is present in an amount of about 0.1%
to 1%; and the indicator agent is present in an amount of about 0.2% to 0.7%. In some
embodiments, the ion-balance reagent is a quaternary ammonium ion. In some
embodiments, the ion-balance reagent is a quaternary ammonium salt.
In some embodiments, the ion-balance reagent is selected from the group
consisting of: di(long-chain aikyl)dimethyl ammonium chloride, N -methyl-N,N -bis(longchain
alkanoyloxyethyl)-N -(2-hydroxyethyl) ammonium methylsulfate,
vinylbenzyldimethylcocoammonium chloride, methyl trioctyl ammonium chloride
tricaprylylmethyl ammonium chloride, tridodecylmethyl ammonium chloride and
cetyltimethyl ammonium chloride. Each possibility represents as separate embodiment of
the present invention.
Any suitable ion-balance reagent may be used, including, tridodecylmethyl
ammonium chloride (TDMAC; CAS 7173-54-8) or cetyltimethyl ammonium chloride
(CTAC; CAS 112-02-7). In some embodiments, the ion balance reagent is a combination
of ion-balance reagents. In some embodiments, the ion-balance reagent is TDMAC
(Tridodecylmethylammonium chloride) .
In some embodiments, the indicator is selected from the group of chemical
compounds having negatively charged functional groups. In some embodiments, the
indicator is a weak acid. In some embodiments, the indicator is selected from the group
consisting of nitrazine yellow, thymol blue, bromthymol blue, xylenol blue, bromoxylenol
blue, phenol red, m-cresol purple, chlorophenol red, bromcresol purple, alizarin, neutral
red, and cresol red. Each possibility represents as separate embodiment of the present
invention. A list of other suitable indicators can be found, for example, in U.S. Patent No.
5,897,834. It is clear to one skilled in the art that the indicators specifically mentioned
herein are just examples and any suitable indicators may be used.
In some embodiments, the indicator agent is selected from the group consisting of:
cresol red, alizarin, bromocresol purple, chlorophenol red, nitrazine yellow, bromthymol
blue, bromoxylenol blue, neutral red, phenol red, thymol blue, xylenol blue and m-cresol
purple. Each possibility represents as separate embodiment of the present invention.
In some embodiments, the indicator agent is negatively charged.
In some embodiments, the indicator agent is a weak acid.
In some embodiments, the indicator agent comprises nitrazine yellow.
In some embodiments, the diagnostic composition is associated with an absorbent
material, thereby forming a diagnostic article for absorbing the fluids secreted from a
pregnant woman. The diagnostic article can be implemented using many devices and
methods. In some embodiments, the article is implemented in a manner that can be easily
used by non-skilled personnel, specifically a user. The body of the article comprising the
absorbent material can be supplied to the user, for example, in the form of a pad, gauze, a
swab, a fiber ball, but most preferably, as a sanitary napkin, diaper, panty shield, and
interlabial structure.
In some embodiments, the article further comprises a means for mounting the
article to facilitate the collection of the secreted biological fluid. An example of a
mounting means includes, but is not limited to, adhesive strips associated with the article.
In some embodiments, the article further comprises release tapes, each covering each of
the adhesive strips. Covering as used herein also means protecting (e.g. from drying out).
Typically, the user removes the release tapes to expose the adhesive strips of the article
and places the article in the crotch portion of their undergarment, thereby preventing the
article from moving out of position during regular use. Types of adhesive compounds that
can be used are well known in the art.
In some embodiments, the diagnostic composition provides a visible indication of
amniotic fluid. In some embodiments, the visual indication is stable for about a week.
The terms "stable indication" and "irreversible indication" are interchangeably
used herein to describe an indication, typically a color indication, that once obtained
remains sufficiently unaltered for the time required for clinical examination by a
professional. Preferably, the color change is stable for at least 48 hours, more preferably at
least 72 hours, and in some embodiments, preferably the color change is stable for about a
week.
In some embodiments, the absorbent material is selected from the group consisting
of a swab, gauze, panty shield, hygienic napkin, diaper and interlabial absorbent structure.
Each possibility represents as separate embodiment of the present invention.
In some embodiments, there is provided a kit comprising the article for detecting
amniotic fluid in vaginal secretion, and instructions for use of the article in order to detect
amniotic fluid in vaginal secretion.
In some embodiments, the kit further comprises a color scale presenting the color
expected to appear upon contact of the article with amniotic fluid and the color expected
to appear upon contact of the article with urine.
In some embodiments, the kit comprises a plurality of article for detecting amniotic
fluid in vaginal secretion.
In some embodiments, each article within the kit is separately wrapped.
The above disclosure generally describes the present invention. A more complete
understanding can be obtained by reference to the following specific Examples. These
Examples are described solely for purposes of illustration and are not intended to limit the
scope of the invention. Changes in form and substitution of equivalents are contemplated
as circumstances may suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and not for purposes of
limitation.
EXAMPLES
EXAMPLE 1: Clinical Study
Samples of urine and amniotic fluid were collected from 83 pregnant women,
according to the following distribution: urine and amniotic fluid were collected from 8
women, urine only was collected from 63 women, and amniotic fluid was collected from
12 women.
The inclusion criteria for subject participation in the study are pregnant women:
) . Pregnant women 18 years to 50 years of age.
(ii). Subject is ready to sign the informed consent form.
(iii). Subject arrives at delivery room undergo amniotomy.
The exclusion criteria for subject participation in this study are:
(iv). Subject suffers from bleeding.
(v). Subject is unable or unwilling to cooperate with study procedures.
(vi). Subject is currently participating in another clinical study.
Subjects arriving at the delivery room were requested to collect a random urine
sample in a clean container. Amniotic fluid samples were collected, by the clinician,
during the amniotomy procedure. Each fresh urine and amniotic fluid sample in the study
was tested using the test devices (panty liners) each comprising candidate formulation or a
reference formulation, as follows:
1. Dipstick for pH and other urine parameters (using Combur-Test® strips, Roche
Diagnostics).
2. of amniotic fluid was dropped onto each device candidate and reference
devices (AL-SENSE and AmniScreen).
3. 4001 of urine was dropped onto each device candidate (a panty liner containing
one of the eight candidate formulations) and each reference device (a panty liner
containing the AmniScreen or Al-Sense formulations).
4. The study coordinator stirred each panty liner in a way that will mimic the "real
life" usage.
5. For the amniotic fluid and urine samples, the coordinator recorded the result status
(positive or negative) immediately (0-2 min), and after 5 min, 10 min, 15 min, 20 min,
30 min, 60 min and 120 min, based on the color observed on the tested pads. A
positive result is concluded when the yellow panty liner show a blue, green stain of any
size or shape, even if as a light color. A negative result is concluded when the panty
liner color remained yellow.
Eight types of device candidates (Table 1) and two reference devices, AL-SENSE
and AmniScreen (Table 2), were used. The ingredients in each candidate and reference
formulations were dissolved in 50 ml acetone and 2 ml water.
Table 1: Formulations of candidate devices
Agent ALMI/ALMIG ALM2/ALM2G ALM3/ALM3G ALM4/ALM4G
Wt. (g) wt/wt(%) Wt. (g) wt/wt(%) Wt. (g) wt/wt(%) Wt. (g) wt/wt(%)
Cellulose acetate 2 44.6 2 45.7 2 45.7 2 45.8
Cellulose acetate- 0.8 17.9 0.8 18.3 0.85 19.4 0.8 18.3
butyrate
Triethyl citrate 1 22.3 1 22.8 1 22.8 1 22.9
TDMAC 0.053 1.2 0.053 1.2 0.053 1.2 0.046 1.1
Poloxamer 0.3 6.7
Salicylic acid 0.3 6.7 0.25 5.71
Benzoic acid 0.3 6.7 0.3 6.86
Tween 80 0.2 6.7 0.2 4.57 0.2 4.58
Nitrazine yellow 0.024 0.54 0.024 0.55 0.024 0.55 0.024 0.55
Total 4.48 100 4.38 100 4.38 100 4.37 100
¾G = contains glue between the layers as a part of the manufacturing process.
Table 2 : AL-SENSE and AmniScreen Compositi ions
Ingredient AL-Sense AmniScreen Units
Cellulose Acetate 1.6 1.5 Gr
Cellulose acetate butyrate 0.8 Gr
Dibutyl phthalate 1.07 Ml
Hexamoll Dinch 0.8 Ml
TDMAC 0.0453 Gr
Aliquat 336 0.23 Ml
2-ethoxyethanol 1.5 Ml
Citric acid 0.01 12 Gr
Nitrazine Yellow 0.0091 0.024 Gr
The values of the AmniScreen reference device in terms of sensitivity and specificity
were as follows: efficacy of 84% specificity and 96% sensitivity. AmniScreen provides
final reading after 30 minutes. Significance level of findings was determined to be equal
to, or lower than, 5%. All statistical analyses were performed using SAS v9.3 (SAS®,
SAS Institute Cary, NC USA) software.
Results (stains) were detected at different time points. While accurate detection with
100% sensitivity was obtained by articles comprising the indicator composition of the
invention within the first 2 minutes after contact with amniotic fluid (Table 3), it was
required to wait 30 minutes and 10 minutes when using AmniScreen and AL-SENSE,
respectively, in order to avoid false positive readings (i.e. staining by urine). This is
because in AmniScreen and AL-SENSE a stain is obtained from non-specific staining (by
urine), which requires some time (30 minutes in AmniScreen and 10 minutes in ALSENSE)
to fade.
The results from the different tests, based on the pads color, were designated
"Positive", "Negative". The term "Positive" means a blue stain, which appeared upon the
yellow background of the panty liner device. The term "Negative" means that a stain did
not appear, or appeared and faded back to yellow during detection time. A stain formed in
a urine test was considered as false positive and when a stain does not appear in a urine
test it was true negative. Similarly, when a stain did not appear in a test of amniotic fluid it
was referred to as false negative.
Tables 3-4 and Figure 1 present the detected results read from the candidate devices
following exposure to amniotic fluid (Table 3) or to urine (Table 4). Efficacy evaluation
was obtained by comparing the sensitivity, i.e. the proportion of "Positive" readings upon
exposure of the pads to amniotic fluid, and the specificity, i.e. the proportion of
"Negative" upon exposure of the pads to urine, from the various devices (pads). The
resulting sensitivities and specificities are presented in Tables 3 and 4 along with the
respective two-sided 90% exact Confidence Intervals (CI) compared with McNemar' s test.
For each candidate device agreement between the candidate device and the AmniScreen
was calculated together with 95% exact confidence intervals. Agreements in amniotic
fluid, in urine and altogether were evaluated as the percentage of equal diagnoses
(positive-positive negative-negative) between pairs of devices. The devices diagnosis
agreement levels were cross-tabulated against those of the AmniScreen overall agreement
levels and compared using McNemar' s test. As shown in Table 3 all candidate devices had
a sensitivity of 100%, i.e. all candidates exhibited a positive reading (blue stain) following
exposure to amniotic fluid. Furthermore, in all candidate pads the color change (blue stain
formation on a yellow background) occurred immediately or by no longer than two
minutes after contacting the test sample (i.e. 0-2 min) and remained stable for the duration
of the two-hour follow-up. Regarding specificity, candidate pads ALM1 and ALM1G
demonstrated superiority over the remaining candidate pads (Table 4 and Figure 1). These
two candidates exhibited 84% specificity at 5 minutes and even better specificity values at
10 minutes with no fluctuations: Specificity of the ALM1 (and ALM1G) candidate at 5
minutes was 84.5% (60/71) and at 10 minutes 87.2% (62/71). Thus, ALMl and ALMIG
meet the goal of good accuracy in a short time span.
Tables 5 and 6 present the sensitivity and specificity, respectively, of five selected
candidate pads, at 5 and 10 minutes along with the results of AL-SENSE and AmniScreen
with lower two-sided 90% exact confidence bounds, based on the values presented in
Tables 3 and 4. According to Table 5, the sensitivity of ALMl at 5 and 10 minutes is
100% (namely, all amniotic fluid samples that were contacted with ALMl, resulted in a
positive detection - blue stain). In terms of performance, it may be stated that for this
study, the value of 86.09% has 95% confidence. Stated otherwise, the value of 86.09%
refers to the assertion that may be made given the sample size and the sensitivity data. The
same applies to AmniScreen. AL-SENSE has a 90% sensitivity, which is lower than that
of ALMl and AmniScreen.
Table 3 : Readings of candidate devices upon exposure to amniotic fluid
Candidate 0-2m 5m 10m 15m 20m 30m 60m 120m
device N % N % N % N % N % N % N % N %
ALMl
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALM2
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALM3
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALM4
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALMIG
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALM2G
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALM3G
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
ALM4G
Positive 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0 20 100.0
'%' -% sensitivity; 'N' - the number of samples tested; 'm' - minutes.
According to Table 6, specificity of ALMl at 5 minutes (84.51%) is better than that
of AmniScreen (77.46%; p=0.0588) with a significant superiority at 10 minutes (87.32%)
relative to AmniScreen (p=0.0082) and AL-SENSE (74.65%, p=0.0067). Specificities of
AmniScreen and AL-SENSE were not significantly different (p=0.5637).
Specificity, as used herein refers, to correct negative reading, namely, non-staining
of a composition upon contact thereof with a urine sample.
The clear and sharp detection with ALMl is further demonstrated in Figures 3A and
3B. Figure 3A presents the dark stain obtained after contacting the composition of ALMl
absorbed into a panty shield with amniotic fluid (the dark stain is surrounded by a dashed
line and an arrow is pointing at the stain). The dark stain presented in Figure 3A is clearer
and much more pronounced compared to the stains observed after contacting the reference
compositions, namely, AL-SENSE and AmniScreen, with amniotic fluid. Figure 3B
presents a corresponding composition of ALMl absorbed into a panty shield after being in
contact with urine. As can be seen, no staining is observed indicating the absence of
amniotic fluid.
Table 4 : Readings of the candidate devices when exposed to urine, over time
0-2m 5m 10m 15m 20m 30m 60m 120m
N % N % N % N % N % N % N % N %
ALMl
Negative 58 81.7 60 84.5 62 87.3 62 87.3 62 87.3 64 90. 1 64 90. 1 64 90. 1
Positive 13 18.3 11 15.5 9 12.7 9 12.7 9 12.7 7 9.9 7 9.9 7 9.9
ALM2
Negative 53 74.6 57 80.3 60 84.5 60 84.5 6 1 85.9 62 87.3 62 87.3 62 87.3
Positive 18 25.4 14 19.7 11 15.5 11 15.5 10 14. 1 9 12.7 9 12.7 9 12.7
ALM3
Negative 52 73.2 56 78.9 60 84.5 6 1 85.9 6 1 85.9 64 90. 1 64 90. 1 64 90. 1
Positive 19 26.8 15 21. 1 11 15.5 10 14. 1 10 14. 1 7 9.9 7 9.9 7 9.9
ALM4
Negative 57 80.3 60 84.5 60 84.5 6 1 85.9 60 84.5 6 1 85.9 6 1 85.9 6 1 85.9
Positive 14 19.7 11 15.5 11 15.5 10 14. 1 11 15.5 10 14. 1 10 14. 1 10 14. 1
ALM1G
Negative 58 81.7 60 84.5 62 87.3 63 88.7 63 88.7 65 91.5 65 91.5 65 91.5
Positive 13 18.3 11 15.5 9 12.7 8 11.3 8 11.3 6 8.5 6 8.5 6 8.5
ALM2G
Negative 56 78.9 59 83. 1 6 1 85.9 6 1 85.9 6 1 85.9 63 88.7 63 88.7 63 88.7
Positive 15 21. 1 12 16.9 10 14.1 10 14. 1 10 14. 1 8 11.3 8 11.3 8 11.3
ALM3G
Negative 56 78.9 59 83. 1 6 1 85.9 62 87.3 63 88.7 64 90. 1 64 90. 1 64 90. 1
Positive 15 21. 1 12 16.9 10 14.1 9 12.7 8 11.3 7 9.9 7 9.9 7 9.9
ALM4G
Negative 56 78.9 58 81.7 60 84.5 60 84.5 6 1 85.9 63 88.7 63 88.7 63 88.7
Positive 15 21. 1 13 18.3 11 15.5 11 15.5 10 14. 1 8 11.3 8 11.3 8 11.3
'%' - % sensitivity; 'N' - the number of samples tested; 'm' - minutes.
Table 5 : Sensitivity of the candidate and reference pads
Device n/N Sensitivity (%) Lower 90% CL
ALMl 5 min 20/20 100.0% 86.09%
ALMl 10 min 20/20 100.0% 86.09%
AL-Sense 10 min 18/20 90.0% 71.74%
AmniScreen 30 min 20/20 100.0% 86.09%
Table 6 : Specificitv of the candidate and reference pads
Device n/N Specificity Lower P-Value vs. P-Value vs.
(%) 90% CL AmniScreen AL-SENSE
ALMl 5 min 60/71 84.51% 75.66% 0.0588 0.0196
ALMl 10 min 62/71 87.32% 78.92% 0.0082 0.0067
AL-SENSE 10 min 53/71 74.65% 64.76% 0.5637 NR
AmniScreen 30 min 55/71 77.46% 67.81% NR 0.5637
The clear and sharp detection with ALMl is further demonstrated in Figures 3A and
3B. Figure 3A presents the dark stain obtained immediately after contacting the
composition of ALMl absorbed into a panty shield with amniotic fluid (the dark stain is
surrounded by a dashed line and an arrow is pointing at the stain). The dark stain presented
in Figure 3A is clearer and much more pronounced compared to the stains observed after
contacting the reference compositions, namely, AL-SENSE and AmniScreen, with
amniotic fluid. Figure 3B presents a corresponding composition of ALMl absorbed into a
panty shield after being in contact with urine. As can be seen, no staining is observed
indicating the absence of amniotic fluid.
Table 7 presents cross-tabulation readings of ALMl and AL-SENSE along with the
reading of AmniScreen - all readings were obtained on the same sample.
Table 8 displays the total percent agreement level of the candidate and the reference
(AL-SENSE and AmniScreen) devices on positive and negative cases and overall with
two-sided 95% exact confidence intervals for each. These values represent are the percent
of cases in which the devices gave the same reading in amniotic fluid (positive cases;
N=20), in urine (negative cases; N=71) and overall (together in both urine and amniotic
fluid; N=91). As can be seen in the positive cases, i.e. amniotic fluid, ALMl readings at 5
and 10 minutes correspond to AmniScreen readings. Thus, the agreement between ALMl
and AmniScreen in diagnosis of amniotic fluid is 100% for both time points, where
agreement between ALMl and AL-SENSE was only 90% (Table 5). In urine, ALMl at 5
minutes agrees with AmniScreen in 98.2% of the cases and at 10 minutes in all (100%)
cases where the AmniScreen provided a negative reading. AmniScreen demonstrated six
false positive readings at 5 minutes and seven false positive readings at 10 minutes while
ALMl correctly read negative reading at 5 minutes and 10 minutes (Table 6).
Table 7 : Cross tabulation of candidate, AL-Sense versus AmniScreen
AmniScreen
Test Sample Negative Positive
N % N %
Amniotic ALMl 5m
Fluid Positive 20 100.0
ALMl 10m
Positive 20 100.0
AL-SENSE
Negative 2 10.0
Positive 18 90.0
Urine ALMl 5m
Negative 54 98.2 6 37.5
Positive 1 1.8 10 62.5
ALMl 10m
Negative 55 100.0 7 43.8
Positive 9 56.3
AL-SENSE
Negative 48 87.3 5 31.3
Positive 7 12.7 11 68.8
Overall ALMl 5m
Negative 54 98.2 6 16.7
Positive 1 1.8 30 83.3
ALMl 10m
Negative 55 100.0 7 19.4
Positive 29 80.6
AL-SENSE
Negative 48 87.3 7 19.4
Positive 7 12.7 29 80.6
In general, there was a disagreement between ALMl at 5 min and lOmin and
AmniScreen in seven cases out of which ALMl gave the correct reading in six (6) cases at
5 min and in all seven (7) cases at the 10 min evaluation point. The differences were not
statistically significant at 5 minutes (p=0.0588, Table 8) but were found statistically
significant at 10 min (p=0.0082, Table 8). Stated otherwise, as evident from Table 7, the
aforementioned disagreement is only on the negative cases (i.e., urine) where at 5 min
ALMl gave the correct reading in 6 of those 7 discrepant cases and in all 7 cases at the 10
min evaluation point. Disagreement between AL-SENSE and AmniScreen was greater -
14 cases, of which the AmniScreen was correct in 9 of the cases. Despite the better
accuracy, AmniScreen and AL-Sense were found to be similar (p=1.00, Table 8) with
respect to percent of positive and negative readings yet less effective than ALMl.
Table 8: Agreement of ALMl and AL-SENSE with AmniScreen
P-Value
Agreement Lower Upper 95% McNemar's
Device n/N Percent 95% CL CL Test
Amniotic ALMl 20/20 100.0% 83.16% 100.0% -
Fluid 5 min
ALMl 20/20 100.0% 83.16% 100.0% -
10 min
AL-Sense 18/20 90.0% 68.30% 99.98% -
Urine ALMl 64/71 90.14% 80.74% 95.94% -
5 min
ALMl 64/71 90.14% 80.74% 95.94% -
10 min
AL-Sense 59/71 83.10% 72.34% 90.95% -
Overall ALMl 84/91 92.31% 84.79% 96.85% 0.0588
5 min
ALMl 84/91 92.31% 84.79% 96.85% 0.0082
10 min
AL-Sense 77/91 84.62% 75.54% 91.33% 1.000
EXAMPLE 2: Clinical Study with Articles Lacking Surfactants and Acids
Two formulations, 3Marl and 3Mar2, were prepared, containing all of the
ingredients of the ALMl formulation aside from poloxamer 407 or salicylic acid (Table
9).
Table 9 : Formulations Lacking Surfactants or Acids
Ingredient ALMl 3Marl 3Mar2
Cellulose Acetate 2.00 2.50 2.50
Cellulose acetate butyrate 0.80 1.00 1.00
Triethyl citrate 1.00 1.00 1.00
TDMAC 0.053 0.053 0.053
Poloxamer 407 0.30 0.30
Salicylic acid 0.30 0.30
Nitrazine Yellow 0.024 0.024 0.024
Urine samples (400 ΐ) having pH 7.0 were dripped on articles containing
the3Marl and 3Mar2 formulations and on an article containing the ALMl formulation.
As shown in Figure 2, following contact with urine each of the articles containing
the 3Marl or the 3Mar2 formulations produced a false positive result, namely, a dark
distinctive (blue) stain formed at the center of each of the articles. However, the ALMl
article provided a true negative result as it did not produce any stain upon contacting the
urine sample.
The results indicate that surfactant(s) and acid(s) are required for obtaining a
reliable detection.
EXAMPLE 3: Feasibility of Wearing Study
A primary efficacy analysis with an article which includes the diagnostic
composition ALMl (also termed hereinafter "AL SENSE Blue") was conducted on a total
of 43 women who were compliant with the study protocol and had valid data for the
analysis. The analysis consists of a comparison between the patient's reading of the ALSENSE
Blue result and the clinical diagnosis.
Table 10 presents the subject's reading of the AL-SENSE Blue device versus the
clinical diagnosis (sterile speculum Pooling test, if result was negative, then a microscopic
Ferning test and pH test were performed).
Table 10: Subject's reading vs. clinical diagnosis
Clinical Diagnosis AL-SENSE Blue Subject's reading
Frequency (N) Positive Negative Total
Negative 1 16 17
Positive 25 1 26
Total 26 17 43
The sensitivity observed in this study is 96.15% (25/26) with a 95% exact binomial
confidence interval of 80.36% - 99.90% and the specificity is 94.12% (16/17) with a 95% exact
binomial confidence interval of 71.31% - 99.85%. Since the Success Criteria were 91% for the
sensitivity and 73% for the specificity, it is concluded that the study was successful.
In a Secondary Efficacy study the women (all participants) were requested to complete a
questionnaire. The first three questions were rated on a scale of 1 through 5, where 1 represents a
poor result and 5 a very satisfactory result. The last two questions relate to patient's
comprehension of the results reading. Tables 11-14 present in a descriptive manner the
distributions of response gathered.
Table 11: Responses to Question 1
Is the Blue-Green Stain on the Indicator Strip
Clear to Distinguish? (N=35)
Response Frequency %
Slightly Clear 1 2.86
Somewhat Clear 10 28.57
Clear 17 48.57
Very Clear 7 20
Table 12: Responses to Question 2 :
Is the pad comfortable for use? (N=40)
Response Frequency %
Quite Comfortable 24 60
Very Comfortable 6 4
Table 13: Responses to Question 3
Did you experience any discomfort related to use of the pad?
(N=41)
Response Frequency %
Indifferent 17 41.46
I was quite comfortable with the ALSENSE
Blue 11 26.83
I experienced no discomfort related to the
AL-SENSE Blue 13 31.71
Table 14: Responses to Question 4
According to the color of the used indicator strip and to the instructions
leaflet, do you understand the cause of your wetness sensation? (N=41)
Response Frequency %
Amniotic fluid
leakage 23 53.66
Urine leakage 0 0
Nothing 7 17.07
I don't know 11 29.27
Of the 35 women that responded to question 1, 97.14% (34/35) responded that the Blue or
green stain on the yellow strip was somewhat clear, clear or very clear to distinguish.
Of the 40 women that responded to question 2, 100% (40/40) responded the pad was
quite or very comfortable for use.
Of the 4 1 women that responded to question 3, no one claimed that they had experienced
even slight discomfort relating to the use of the AL-SENSE Blue.
Of the 4 1 women that responded to question 4, 24 marked their reading as positive.
95.83% of these women (23/24) stated that from the strip they had understood that the cause of
their wetness was due to amniotic fluid leakage, all of them actually marked their reading as
positive.
'Time to Reading' of the tested article was improved relative to the reference articles.
Specifically, reading of the liner color was detected and recorded at 2, 5, 10, 15, 20 and 30
minutes (Table 15). In 100% (43/43) of the cases the final reading was already present from 2
minutes after exposition. In contrast, the Time to Reading of the reference article AmniScreen
is 30 minutes while the Time to Reading of the reference article AL-SENSE is at least 10
minutes.
Table 15: Listing of the readings over time
Patient Patient patient Patient Patient Patient
Subject
result result result result result result
Number
0-2 min 5 min 10 min 15 min 20 min 30 min
9200 Positive Positive Positive Positive Positive Positive
9201 Positive Positive Positive Positive Positive Positive
9202 Positive Positive Positive Positive Positive Positive
9203 Negative Negative Negative Negative Negative Negative
9204 Positive Positive Positive Positive Positive Positive
9205 Positive Positive Positive Positive Positive Positive
9206 Negative Negative Negative Negative Negative Negative
9207 Positive Positive Positive Positive Positive Positive
9208 Positive Positive Positive Positive Positive Positive
9209 Positive Positive Positive Positive Positive Positive
9210 Positive Positive Positive Positive Positive Positive
9212 Negative Negative Negative Negative Negative Negative
9213 Negative Negative Negative Negative Negative Negative
9214 Negative Negative Negative Negative Negative Negative
9215 Negative Negative Negative Negative Negative Negative
9217 Positive Positive Positive Positive Positive Positive
9218 Positive Positive Positive Positive Positive Positive
9219 Positive Positive Positive Positive Positive Positive
9222 Positive Positive Positive Positive Positive Positive
9223 Positive Positive Positive Positive Positive Positive
9224 Positive Positive Positive Positive Positive Positive
9226 Negative Negative Negative Negative Negative Negative
9227 Positive Positive Positive Positive Positive Positive
9228 Negative Negative Negative Negative Negative Negative
9229 Negative Negative Negative Negative Negative Negative
9230 Positive Positive Positive Positive Positive Positive
9231 Positive Positive Positive Positive Positive Positive
9232 Positive Positive Positive Positive Positive Positive
9233 Negative Negative Negative Negative Negative Negative
9234 Negative Negative Negative Negative Negative Negative
9237 Positive Positive Positive Positive Positive Positive
9238 Negative Negative Negative Negative Negative Negative
9239 Negative Negative Negative Negative Negative Negative
9240 Negative Negative Negative Negative Negative Negative
9241 Negative Negative Negative Negative Negative Negative
9242 Positive Positive Positive Positive Positive Positive
9243 Negative Negative Negative Negative Negative Negative
9244 Negative Negative Negative Negative Negative Negative
9246 Positive Positive Positive Positive Positive Positive
9247 Positive Positive Positive Positive Positive Positive
9248 Positive Positive Positive Positive Positive Positive
7189 Positive Positive Positive Positive Positive Positive
7190 Positive Positive Positive Positive Positive Positive
In summary of the primary and secondary studies, the AL-SENSE Blue subject reading
sensitivity is 96.15%, which is very good for a home use test. Furthermore, the performance
altogether (sensitivity, specificity, time of reading etc.) of AL-SENSE Blue is better than that of
the reference articles AL-SENSE and AmniScreen.
EXAMPLE 4: Article Preparation
Article preparation initially involved preparing each of the candidate and reference
(including 3Marl and 3Mar2) formulations in the form of solutions. For this purpose, the
solid part of each formulation, excluding Nitrazine yellow, was dissolved in 50 ml
acetone. Nitrazine yellow was dissolved in 2 ml water and then added to the acetone
solution.
The resulting solutions were sprayed on a substrate material. As a result, the
substrate material acquired a faint yellow color, which gave a suitable background for the
detection of a color change due to interaction with vaginal fluids, particularly, for the
detection of a blue stain as a proper result of interaction with amniotic fluid. Each
substrate was then integrated with a pre -formed panty liner, thereby obtaining a distinct
diagnostic (candidate or reference) panty liner.
The foregoing description of the specific embodiments will so fully reveal the
general nature of the invention that others can, by applying current knowledge, readily
modify and/or adapt for various applications such specific embodiments without undue
experimentation and without departing from the generic concept, and, therefore, such
adaptations and modifications should and are intended to be comprehended within the
meaning and range of equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the purpose of description and
not of limitation. The means, materials, and steps for carrying out various disclosed
functions may take a variety of alternative forms without departing from the invention.
CLAIMS
1. An indicator composition for detecting amniotic fluid in vaginal secretion
comprising: a plurality of polymers; a non-cyclic plasticizer; a hydrophilic
surfactant; an aromatic carboxylic acid; an ion-balance reagent and an indicator
agent, wherein said composition is devoid of a wetting agent.
2. The indicator composition of claim 1, wherein said aromatic carboxylic acid
comprises salicylic acid, benzoic acid or a combination thereof.
3. The indicator composition of claim 1, wherein said aromatic carboxylic acid is
salicylic acid.
4. The indicator composition of claim 1, wherein said hydrophilic surfactant is a nonionic
surfactant.
5. The indicator composition of claim 1, wherein said hydrophilic surfactant is a
polymer.
6. The indicator composition of claim 5, wherein said hydrophilic surfactant comprises
a block copolymer surfactant.
7. The indicator composition of claim 1, wherein the indicator agent is negatively
charged.
8. The indicator composition of claim 1, wherein the ion-balance reagent is a
quaternary ammonium salt.
9. The indicator composition of claim 1, wherein the non-cyclic plasticizer is a tri-ester
plasticizer.
10. The indicator composition of claim 1, consisting of a plurality of polymers; a noncyclic
plasticizer; a hydrophilic surfactant; an aromatic carboxylic acid; an ionbalance
reagent and an indicator agent.
11. An article for detecting amniotic fluid in vaginal secretion comprising: an absorbent
material for absorbing a biological fluid secreted from a person, and the indicator
composition of any one or more of claims 1-10.
12. The article of claim 11, wherein the absorbent material is selected from the group
consisting of a swab, gauze, panty shield, hygienic napkin, diaper and interlabial
absorbent structure.
13. A method for diagnosing amniotic fluid leakage in vaginal secretion comprising:
(d) positioning a secretion-monitoring article to receive a biological
fluid secreted from the subject, the article comprising the indicator composition of
claim 1;
(e) contacting said article with vaginal secretion; and
(f) viewing the article, wherein a color change indicates the presence of
amniotic fluid.
14. The method of claim 13, wherein said color change is a dark stain relative to
background of the secretion monitoring article.
15. The method of claim 14, wherein the dark stain is a dark blue stain or a dark green
stain.
16. The method of claim 13, wherein said viewing is performed within less than 10
minutes after said contacting.
17. The method of claim 16, wherein said viewing is performed within less than 5
minutes after said contacting.
18. The method of claim 13, wherein said indicator composition is consisting of a
plurality of polymers; a non-cyclic plasticizer; an hydrophilic surfactant; an aromatic
carboxylic acid; an ion-balance reagent and an indicator agent.
19. A kit comprising the article of claim 11 for detecting amniotic fluid in vaginal
secretion, and instructions for use.
20. The kit of claim 19, further comprises a color scale presenting the color expected to
appear upon contact of the article with amniotic fluid.