characterisation of poly vinyl alcohol
TRANSCRIPT
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Characterisation of Poly vinyl alcohol (PVA) / Silver nitratenanomembranes for their suitability in wound dressing
applicationsDr. K. P. Chellamani1, P. Sundaramoorthy2, T. Sureshram3
1,2,3The South India Textile Research Association (SITRA), Coimbatore641 014,
Tamil Nadu, India
Abstract - Special wound care product was developed using
Poly vinyl alcohol (PVA) blended with Silver nitrate.
Electrospinning technique was adopted to produce
PVA/Silver nitrate nanomembranes, which was used as
wound dressings. PVA/Silver nitrate wound dressings(PSNWD) have high moisture vapour transmission property
and good antimicrobial activity. PSNWD substrates have mild
cytotoxicity reactivity and have excellent odour absorbing
capability. PVA/Silver nitrate wound dressing do not cause
skin irritation even after 72 hours of contact with the wound
and the time taken for wound healing while using PSNWD is
just 50% of that in the case of an open wound.
Keywords- Cytotoxicity, Electrospinning, nanomembranes,
Poly vinyl alcohol (PVA), Silver nitrate and wound dressing.
I. INTRODUCTION Electrospinning [1] is a technique used to process
polymer fiber diameter in the range of micrometers tonanometers. Electrospinning process has increased in
recent years, and this technology has been exploited for a
wide range of applications. Among various nanomembrane
processing techniques like drawing, template synthesis,
phase separation and self assembly, electrospinning is the
only method capable of producing continuous polymer
nanofibres. Electrospun polymer nanofibers have very large
surface area to volume ratio and flexibility in surface; these
properties make the polymer nanofibers use in many
important applications. Electrospun nanofibers achieve
good result on properties required for wound dressings,
because of its nano-size structure.
Choice of wound dressings [2] is dependent upon the
severity and type of injury, as well as the stage of healing
and the presence or absence of infection. The essential
parameters require for wound dressing to heal wounds are
Absorptivity [3] and oxygen permeability [3]. Additionally,
ability to conform to the wound bed, provide appropriate
adherence/non-adherence to the healing tissue [4], provide
a barrier to bacteria, enhanced moisture management5,
bioactivity, resorbability [5] and occlusivity are highly
desirable and correlated with better outcomes.
Poly (vinyl alcohol) (PVA) has excellent chemical
resistance, physical properties, biodegradability and good
fiber forming capability in electrospinning techniques [6].
PVA polymer is suitable for blending various polymers and
additives in electrospinning process to achieve uniformnanofibers [7].
*Correspondence to the author:
P. Sundaramoorthy
E-Mail ID: [email protected]
Silver nitrate is a salt used to produce Silver
nanoparticles by insitu polymerisation with PVA polymer.
Silver nanoparticles have been widely used in various
biomedical fields like wound dressing materials, body wall
repairs, augmentation devices, tissue scaffolds,
antimicrobial filters, and so forth. In the present study, the
PVA polymer is blend with Silver nitrate in different blend
proportions and optimising the electrospinning parameters
using scanning electron microscope (SEM) analysis and
antimicrobial activity [8]. The optimised parameter is used
for further trials and the produced nanomembrane were
characterised for Fourier transform infrared spectroscopy
(FTIR) and transmission electron microscope (TEM)
analysis. The PSNWD is test for wound dressing properties
like Moisture vapour transmission rate (MVTR) [9],
cytotoxicity [10], odour control capability [9], Skin
irritation and wound healing ability
II. MATERIALS AND METHODSA. Materials
The polymers, chemicals and solvents used for this study
are given below
i. Poly Vinyl Alcohol (PVA) with a molecular weightof 1,24,000 g/mol
ii. Silver nitrate saltiii. Nitric acid andiv. Distilled water.
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B. Methods
The electrospinning technique was used to produce thePVA/Silver nitrate nanomembranes. In electrospinning
technique, there are two methods for the preparation of
polymer solution.
1. Method I:In this method, the polymer is dissolved in
the solvent to form polymer solution.
2. Method II:In this method, the polymer is heated upto
its melting point and thereby the polymer solution is
formed.
In the present study, Method I was adopted to prepare
the polymer solution.
C. Solution Preparation
The n gram of polymer is weighed with the help of
weighing balance and m milliliter of solvent is measured
with the help of micropipette.
The concentration of the polymer solution is determined
by the formula,
Concentration =n gram of polymer
X 100 (%)m milliliter of solvent
D. PVA/Silver Nitrate Blend Solution Preparation
The n gram of PVA and y gram of Silver nitrate salt
were dissolved in m millilitre of distilled water (pH of
distilled water - 4).
PVA/Silver nitrate blend solution of 0.5* %, 1 % & 2 %were prepared at 80 C.
* 0.5 % by weight of Silver nitrate in the blend solution of
PVA/Silver nitrate.
E. Electrospinning of Nanomembrane
The term nano represents the size of the material in
10-9
meter. The Polymeric nanofibers can be processed by a
number of techniques like Drawing, Template Synthesis,
Phase Separation, Self-Assembly and Electrospinning.
Among the various processing techniques, electrospinning
is the only method capable of producing continuous
polymer nanofibres. Electrospinning is a unique technology
that can produce non-woven fibrous articles with fiberdiameters ranging from tens of nanometers to microns, a
size range that is otherwise difficult to achieve by
conventional techniques.
A schematic representation of Electrospinning set up is
given in Figure 1. The basic setup for electrospinning
consists of three major components: a high-voltage power
supply, a spinneret (a metallic needle), and a collector (agrounded conductor).
A high electrical supply, typically 130 kV, is applied to
a polymer solution contained in a syringe.
Fig 1 Schematic Diagram of Electrospinning Setup
The electrospinning process is governed by manyparameters, classified broadly into solution parameters,
process parameters, and ambient parameters. Solution
parameters include viscosity, conductivity, molecular
weight, and surface tension. Process parameters include
applied electric field, tip to collector distance and feeding
or flow rate. Ambient parameters include temperature &
humidity maintained during electrospinning process. These
parameters significantly affect the fiber morphology
obtained. By proper manipulation of these parameters,
nanofibers of desired morphology and linear density can be
obtained.
III. PREPARATION AND OPTIMISATION OF PVA/SILVERNITRATE NANOMEMBRANE
PVA/Silver nitrate solution was prepared by In-situ
polymerisation method. In this method, the Silver nitrate
and PVA were mixed in distilled water with pH value at
4.0 (pH value of distilled water maintained by adding
Nitric acid with the same). Maintaining pH value of the
distilled water at 4.0 is mainly to control the ionisation
reaction of Silver ions. To produce PVA/Silver nitrate
nanomembrane, 4 different combinations of PVA/Silver
nitrate were taken. They are,
- 10% PVA/ 0.5*% Silver nitrate- 10% PVA/ 1.0% Silver nitrate- 10% PVA/ 1.5% Silver nitrate and- 10% PVA/ 2.0% Silver nitrate
* 0.5% by weight of Silver nitrate on the weight of PVA
solution with which Silver nitrate is added.
These 4 combinations of PVA/Silver nitrate were
electrospun to form nanomembranes.
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During electrospinning, process parameters like i)
Applied voltage, ii) Flow rate of solution and iii) Tip tocollector distance were optimised by adopting the same
method followed for optimisation of process parameters for
spinning PVA/Chitosan nanomembrane. A total numbers of
27 trials were conducted in the optimisation process.
Optimum values of electrospinning process parameters for
the 4 combinations of PVA/Silver nitrate are given in
Table 1.
Table 1
Optimum Values of Process Parameters* for the PVA / Silver NitrateNanomembranes
S.No.
Combinations
Process parameters
10%
PVA/
0.5%Silver
nitrate
10%
PVA/
1.0%Silver
nitrate
10%
PVA/
1.5%Silver
nitrate
10%
PVA/
2.0%Silver
nitrate
1.Applied voltage
(kV)18 15 18 18
2.Flow rate
(milliliter/hr)0.5 0.5 1.0 0.5
3.Tip to collector
distance (cm)13 8 10 8
*Optimum process parameter in electrospinning is one
where uniform fibres without any bead could be produced.
Figure 2, shows the electrospun PVA/Silver nitrate
nanomembrane obtained after 5 hours of process (21 cm
length).
Fig 2 Electrospun PVA/Silver Nitrate Nanomembrane (after 5 hours
of processing)
SEM picture of PVA/Silver nitrate nanomembrane
produced using different combinations of PVA/Silvernitrate are shown in Appendix I. PVA/Silver nitrate
nanomembrane produced using different blend proportions
were also tested for their antibacterial activity.
A. Antimicrobial Activity of PVA/Silver NitrateNanomembranes
The antimicrobial activity of the nanomembrane is very
important for wound dressing applications. Antimicrobial
test was conducted based on quantitative analysis by JIS L
1902 method. Bacteria used in this study were
staphylococcus aureus (ATCC 6538)*.
*S. Aureus is a Gram positive micro-organism.
Antimicrobial activity of the nanomembranes developedin various percentages of PVA/ silver nitrate viz 10/0.5,
10/1, 10/1.5 and 10/2 was tested as per the standard JIS L
1902. Briefly, 0.2 ml of the Staphylococcus aureus has
been inoculated on 0.5 g of each sterile test material and
incubated for 18 h at 37 C. Following incubation, ratio of
microbiostasis was calculated using the formula F=
Mb -Maand the result was expressed in log reduction. Here
F is growth value, Mb is average value of common
logarithm of number of living bacteria after 18 hr andMais
average of value of common logarithm of number of living
bacteria at 0th
hr. Antimicrobial activity of the PVA/ silver
nitrate nanomembranes and 100% PVA nanomembranes
are given in Table 2.The results confirm that amongvarious combinations of PVA/ silver nitrate, the
nanomembranes developed using 10% PVA/ 2% Silver
nitrate, completely arrested the bacterial growth (100 %)
when compared to control (0 %)*.Hence this combinationhas been chosen for further studies.
Table 2
Antimicrobial Activity of PVA/silver Nitrate Nanomembrane
S.No.
PVA/Silver nitrate
nanomembrane
combinations
% Bacteria reduction
after 18 h of treatment Remarks
Control Treated
1 10% / 0.5% 0 54Moderate
activity
2 10% / 1.0% 0 75Good
activity
3 10% / 1.5% 0 82Goodactivity
4 10% / 2.0% 0 100Very Good
activity
*Control refers to nanomembranes made out of 100% PVA.
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IV. CHARACTERISATION OF PVA/SILVER NITRATENANOMEMBRANE
A. Moisture Vapour Transmission Rate (MVTR) of PVA/Silver Nitrate Nanomembrane
The MVTR is an important criteria for an wound
dressing material. The liquid formed inside the wound layer
first changes to vapour state and then transported to
atmosphere. This moisture vapour transmission helps to
heal the wound; otherwise will be wound infection. The
PVA/Silver nitrate nanomembranes were tested for MVTR
as per BS EN 137262: 2002 [9].
A test sample of 40 mm diameter is taken and fixed over
a container of 35.7 mm inner diameter, containing 20 ml of
distilled water. The test sample container is weighed (W 1)
before the start of the test. Then the container is kept insidean incubator for 24 hours (conditions maintained inside the
incubator i) Temperature: 37 1 C and ii) RH 20%). After
24 hours, the container is taken out and again weighed
(W2).MVTR is calculated based on the formula,
X = (W1W2) 1000 x 24/T
Where,
X is MVTR (g/m2/24 hours)
W1 is the mass of the container, sample and liquid in
grams
W2 is the mass of the container, sample and liquid ingrams after the test duration
and
T is the test period in hours
MVTR values of PVA/ Silver nitrate nanomembrane
samples provided in this study are given in Table 3.
Table 3
MVTR Values of PVA/Silver Nitrate Nanomembranes
S. No.PVA/Silver nitrate
nanomembrane combinations
MVTR (g/m2/24
hours)
1. 10 %/2 % 3060
2. 10%/1.5 % 3070
3. 10%/1.0 % 31204. 10%/0.5 % 3180
With increase in the concentration of Silver nitrate in
PVA, MVTR values tend to increase. This is attribute to
the reduction in the diameter of the corresponding
nanomembrane substrates. For an infected skin, MVTR
value of 2000 to 2500 in good [11]. Hence, all the
PVA/Silver nitrate nanomembrane substrates made in this
study can be considered as suitable for infected skins.
B. Fourier-Transform Infrared Spectroscopy (FT-IR) forPVA/ Silver Nitrate Nanomembrane
Functional groups present in PVA/Silver nitrate
electrospun nanomembranes were studied using FTIR
spectroscopy.
The FTIR spectra of electrospun PVA/ Silver nitrate
nanomembrane (Combination of 10% / 2%) are shown in
Figure 3.
Fig 3 FT-IR Spectra of PVA/Silver Nitrate Nanomembrane
In FTIR spectra, X-axis represents wavenumbers and Y-
axis, the transmittance. It is discernable from the FTIRspectra, that these are peaks at i) Wavenumbers of 3336
cm-1
(peak 1) and ii) wavenumber of range below 500 cm-1
(peak 2). Peak 1 indicates the presence ofOH groups and
peak 2, the presence of silver groups. This inturn confirms
the presence of PVA polymer and nano Silver
materials in the substrates produced.
C. Cytotoxicity Studies on PVA/Silver NitrateNanomembrane
An in vitro cytotoxicity test was conducted for the
PVA/Silver nitrate substrates using Direct contact method
was performed using test sample PVA/Silver nitrate
nanomembrane as per ISO 10993-5 [10].
In this method, a monolayer culture of L-929 is used as
the medium. Test samples, negative and positive controls
were placed on the medium. After incubation at 37 1 C
for 24 hours, the monolayer medium is examined
microscopically for the response around the test specimen.
The reactivity levels observed are graded as 0,1,2,3 & 4
based on zone of lysis, vacuolization, detachment and
membrane disintegration as explained in Table 4.
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Table 4
Grading of Cytotoxicity
Reactivity Description of reactivity zone Grade
NoneNo detectable zone around or under
sample0
SlightlySome malformed or degenerated cells
under specimen1
Mild Zone limited to area under specimen 2
ModerateZone extending specimen size up to 0.33
cm3
SevereZone extended farther than 0.33 cm
beyond specimen4
As per ISO 10993 5, reactivity grade higher than 2 is
considered as cytotoxicity. The PVA/Silver nitratenanomembrane substrates produced in this study (10%/2%)
were found to have Mild reactivity and hence graded as
2 as far as cytotoxicity is concerned.
D. Odour Control Ability of PVA/Silver NitrateNanomembrane
The objective of the test is to assess the resistance of
wound dressing to penetration by odour. The sample is
tested as per BS EN 13726 6: 2003 method [9]. A
stainless steel container of 35.7mm inner diameter and
40mm height is used for carrying out the test. The test
consists of two parts.
1. Part I: Here, the container is heated for 1 hour at 105 C.Then, Nitrogen gas is purged inside the container. After
this, 0.5 l of pure Diethylamine is also passed inside the
container and the container is kept inside anincubator at 37C for 20 minutes. After 20 minutes, 250 l sample of
Nitrogen gas is removed from the container and injected
into GC (Gas Chromatography) instrument. The GC
instrument estimates the intensity ofDiethylamine presentin Nitrogen gas and provides the information in the form of
a graph (Figure 4 a). In the graph, the intensity of
diethylamine present in Nitrogen gas is plotted against
time.
2. Part II: The container is heated for 1 hour at 105 C.
After this, a 1.3% w/v solution of Diethylamine (0.26 gramof Diethylamine dissolved in 20 ml of distilled water) is
added into the container. Then, the test specimen is kept
inside the container, Nitrogen gas is purged inside the
container and the container is kept inside an incubator at 37
C for 30 minutes. After 30 minutes, 250 l sample of
Nitrogen gas is removed from the container and injected
into GC (Gas Chromatography) instrument for the analysis
of intensity of Diethylamine in Nitrogen gas. The
information is given in Figure 4 b.
Fig 4 a) Diethylamine Vs time in Control specimen
Fig 4 b) Diethylamine Vs time in PVA/Silver nitrate nanomembrane
In the Graph, the peak shown at 25 27 minutesrepresents the Diethylamine. The Diethylamine present in
PVA/Silver nitrate nanomembrane is just 5% of that
exhibited by control specimen. Hence, PVA/Silver nitrate
nanomembrane has excellent odour absorbing capability.
E. Skin Irritation Potential of PVA/Silver NitrateNanomembrane
PVA/Silver nitrate nanomembrane samples were
evaluated for potential skin irritation, when they are used
for covering the wound. The evaluation was as per ASTM
F 719-81 standards. The test method is explained in
Appendix II. Six healthy rabbit were selected for the study
and PVA/Silver nitrate wound dressings were applied on
the 6 rabbits. The study has shown that PVA/Silver nitrate
wound dressing do not causes any skin irritation even after
72 hours of contact with the wound.
F. Wound Healing Ability of PVA/Silver NitrateNanomembrane
The extent of wound healing provided by a given wound
dressing was evaluated using the method proposed by
Morton & Malone.
Time (minutes)
IntensityofDiethylamin
e
I
i
i
l
i
Time (minutes)
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As per this method, twelve healthy rats were employed
for the experimentation and they were separated into 2groups (Group I and Group II) each with 6 rats. Excision of
wounds was made on the rats as per the method suggested
by Morton & Malone [12].
The rats were anaesthetized with an aesthetic ether and
placed in operation table in their natural position. A squarewound of about 1.5 cm (width) x 0.2 cm (depth) was made
on depilated ethanol-sterilized dorsal thoracic region of
rats. Infection was made on wounds by staphylococci
aureus.
Table 5
Wound Healing in Open Wound and PVA/Silver Nitrate Nanomembrane Treated Wound
Type of woundsExtent of Wound healing
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
Open wound
PVA / Silver
nitrate
nanomembrane
Group I rats were left as they were withoutapplication of any wound dressing (open wound)
Group II rats were treated using PVA/Silver nitratenanomembrane wound dressing
The dressings were applied on the wounds of the rats
every day till the epithelialisation was complete. The extent
of wound contraction was studied by tracing the raw wound
area in a tracing paper on 0th
day, 7th
day, 14th
day, 21th
day,
28th
day and 35th
day.
The weights of the traced portions of the wounded area
of rats subjected to different treatments were measured
using electronic balance. Based on the difference in weight,
the superiority or otherwise of a particular wound dressing
is determined. Table 5 shows, the extent of wound healing
noticed while using different wound dressings on differentdays.
The weights of the traced portions of open wound and
PVA/ Silver nitrate nanomembrane treated wound ondifferent days are shown in Table It is clear from Table 6,
that there is a decrease in wound area with the application
of PVA/ Silver nitrate nanomembrane wound dressings.
Table 6
Weight Of The Traced Portions Of Wounds On Different Days
Type of
wound
Weight of traced portions of the wound (grams)
Day 0 Day 7Day
14
Day
21
Day
28
Day
35
Open
wound100 154.89
110.7
882.44 40.72
11.2
3
PSNWDtreatedwound
100 61.74 20.87 0
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G. Transmission Electron Microscope (TEM) Analysis ofPVA/Silver Nitrate Nanomembrane
The Transmission electron microscope (TEM) analysis
was used to study the size and distribution of Silver
nanoparticles inside the single PVA/Silver nitrate nanofiber
(Figure 5).
Fig 5 TEM Image of PVA/Silver Nitrate Nanofiber
In the Figure 5, the black dots represent the Silver
nanoparticles. It is clear that the distribution of Silver
nanoparticles on the nanofibre is quite uniform. The
average size of the Silver nanoparticles was found to
be 3.7 nm.
V. CONCLUSIONS SUMMARISING THE ACHIEVEMENTSAND INDICATION OF SCOPE FOR FURTHER WORK
SITRA has developed special type of wound dressingviz) PVA/Silver nitrate nanomembrane wound
dressing (PSNWD).
PSNWD are found to be ideally suited for infectedskins in view of their high moisture vapour
transmission property (MVTR).
PSNWD Substrates have good antimicrobial activity. PSNWD substrates have mild cytotoxicity reactivity
and exhibited excellent odour absorbing capability.
PVA/Silver nitrate substrates do not cause any skinirritation even after 72 hours of contact with thewound.
The time taken for wound healing while usingPSNWD is around 50% lower as compared to that in
the case of an open wound.
REFERENCES
[1] Zheng-Ming Huang et al., 2003. A review on polymer nanofibers byelectrospinning and their applications in nanocomposites. Compos.
Sci. Technol. 63: 2223-2253.
[2] Marian McCord, 2009. Challenges in advanced nanofiber wounddressings (Annual report, National Textile Center). Retrived from
http://ebookbrowse.com/f09-ns06-a9-pdf-d55286555.
[3] Joshua S. et al., 2008. Wound healing dressing and drug deliverysystems: A review. J. Pharm. Sci. 97 (8): 2892-2922.
[4] Zhong S. P. et al., 2010. Tissue scaffolds for skin wound healing anddermal reconstruction. WIREs Nanomedicine and Nanotechnology.
2:510-525.
[5] Mehrdad Kokabi et al., 2007. PVA-Clay nanocomposite hydrogelsfor wound dressing. Eur. Polym. J. 43: 773-781.
[6] Bin Ding, 2002. Preparation and characterisation of nanoscaledPoly(Vinyl alcohol) fibers via electrospinning. Fiber. Polym. 3(2):
73-79.
[7] Kim Soo-Hwan. et al., 2011. Antibacterial activity of Silver-nanoparticles against staphylococcus aureus and Escherichia coli.
Korean J. Microbiol. Biotechnol. 39 (1):77-85.
[8] JIS L 1902: 2008. Testing for antibacterial activity and efficacy ontextile products. Japanese Industrial Standard (JIS). ICS 07.100.99:59.080.01.
[9] BS EN 13726: 2002. Testing methods for primary wound dressings.British Standard. ICS 11.120.20.
[10] ISO 10993-5: 2009. Biological evaluation of medical devices Part5: Tests for in vitro cytotoxicity. International standard.
[11] Ching-Wen Lou, Ching-Wen Lin, Yueh-Sheng Chen, Chun-HsuYao, Zen-Shoung lin, Chieh-Yu Chao and Jia-Horng Lin. 2008.
Dressing properties evaluation of Tencel/Cotton nonwoven fabriccoated with Chitosan for wound. Text. Res. J. 78(3):248-253.
[12] Morton J. J. P. and Malone M. H., 1972. Evaluation of vulneraryactivity by an open wound procedure in rats. Arch. Int. Pharmacod.
T. 196:117-126.
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Appendix I
SEM pictures of PVA/Silver nitrate nanomembrane produced using different combinations of PVA/Silver nitrate
S.
No.
PVA / Silver nitrate
combination
SEM pictures of PVA/ Silver nitrate
nanomembrane
Nanofiber
diameter
(nm)
Results
1. 10% PVA/ 0.5% Silver
nitrate
242 56 Uniform fibers.
No bead
formation.
2. 10% PVA/ 1.0% Silver
nitrate
234 153 Uniform fibers.
No bead
formation.
3. 10% PVA/ 1.5% Silver
nitrate
184 54 Uniform fibers.
No bead
formation.
4. 10% PVA/ 2.0% Silver
nitrate
169 75 Non-uniform
fibers.
Bead formation
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Appendix II
Standard test method for assessing skin irritation
(ASTM F 719-81 standard)
Principle of Measurement
Exposure of skin to the test material is accomplished by
means of a patch test technique employing two intact sites
on the back of each of six albino rabbits. The skin is
clipped free of hair one day prior to testing. The test
substance is applied using 0.5 ml for liquids, 0.5 g for
solids or semisolids and a 2.5 by 2.5 cm square patch for
films. After application, each test site is covered with a 2.5by 2.5 cm gauze flat and entire trunk is occluded with a
polyethylene sleeve. After 24 hours the sleeve, flat and test
material are removed and test sites are evaluated for
erytheme and edema.
Scoring Method
Using the criteria given in Table 7, the test sites are
scored for Erythema (ER) and Edema (ED).
Table 7Skin Reactions Score For Erythema And Edema Formations
Reaction Description ScoreErythema (ER) Erythema and Eschar
No erythema
Very slight erythema (barely perceptible)
Well-defined erythema (pale red in colour)
Moderate to severe erythema (red and area well defined)
Severe erythema (beet redness to slight eschar formation)
0
1
2
3
4
Edema (ED) Edema formation
No edema
Very slight edema (barely perceptible)
Slight edema (edges of area well defined by definite raising)
Moderate edema (edges raising approximately 1 millimeter)
Severe edema (raised more than 1 mm and extending beyond the area of
exposure)
0
1
2
3
4
Test sites can also be scored for erythema and edema at
48 hours as well as 72 hours after removal (as per the usage
requirement) using the criteria given in Table 7.