Mast Cell Dependent Vascular Changes Associated with
an Acute Response to Cold Immersion in Primary Contact
Joseph Meyer1, Alexander M. Gorbach1, Wei-Min Liu1, Nevenka Medic2, Michael Young3,
Celeste Nelson2, Sarah Arceo2, Avanti Desai2, Dean D. Metcalfe2, Hirsh D. Komarow2*
1Infrared Imaging and Thermometry Unit, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, United States
of America, 2Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of
America, 3Clinical Research Directorate/CMRP, SAIC-Frederick, NCI Frederick, Frederick, Maryland, United States of America
Background: While a number of the consequences of mast cell degranulation within tissues have been documented
including tissue-specific changes such as bronchospasm and the subsequent cellular infiltrate, there is little known about
the immediate effects of mast cell degranulation on the associated vasculature, critical to understanding the evolution of
mast cell dependent inflammation.
Objective: To characterize the microcirculatory events that follow mast cell degranulation.
Methodology/Principal Findings: Perturbations in dermal blood flow, temperature and skin color were analyzed using
laser-speckle contrast imaging, infrared and polarized-light colorimetry following cold-hand immersion (CHI) challenge in
patients with cold-induced urticaria compared to the response in healthy controls. Evidence for mast cell degranulation was
established by documentation of serum histamine levels and the localized release of tryptase in post-challenge urticarial
biopsies. Laser-speckle contrast imaging quantified the attenuated response to cold challenge in patients on cetirizine. We
found that the histamine-associated vascular response accompanying mast cell degranulation is rapid and extensive. At the
tissue level, it is characterized by a uniform pattern of increased blood flow, thermal warming, vasodilation, and recruitment
of collateral circulation. These vascular responses are modified by the administration of an antihistamine.
Conclusions/Significance: Monitoring the hemodynamic responses within tissues that are associated with mast cell
degranulation provides additional insight into the evolution of the acute inflammatory response and offers a unique
approach to assess the effectiveness of treatment intervention.
Citation: Meyer J, Gorbach AM, Liu W-M, Medic N, Young M, et al. (2013) Mast Cell Dependent Vascular Changes Associated with an Acute Response to Cold
Immersion in Primary Contact Urticaria. PLoS ONE 8(2): e56773. doi:10.1371/journal.pone.0056773
Editor: Christian Schulz, King’s College London School of Medicine, United Kingdom
Received July 27, 2012; Accepted January 15, 2013; Published February 22, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Division of Intramural Research, NIAID, NIH. Support by M.Y. for this project has been funded in whole or in part with
federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not
necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or
organizations imply endorsement by the U.S. Government. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Mast cell activation and mediator release associated with
allergic inflammation is correlated with a number of immediate
physiologic changes within affected tissues including broncho-
spasm and mucus production in the lungs; abdominal pain
associated with edema and diarrhea in the gastrointestinal tract
and pruritic edema in the skin [1,2,3]. Microscopically mast cell
activation is then followed by influx of inflammatory cells [4,5,6,7].
However in spite of such observations, little is known about the
direct immediate and critical effect of human mast cell degran-
ulation on regional blood flow and the vasculature in the area of
the evolving acute response.
In order to better understand these vascular responses in human
tissues, we chose to initiate mast cell degranulation by a physical
stimulus known to activate mast cells . Mast cell degradation
appears to be the feature that differentiates the consequences of
cold exposure between normal individuals and those with cold
urticaria [9,10]. We therefore designed a clinical study to induce
mast cell degranulation by cold challenge in patients with cold
urticaria (CUrt). To determine the timing and character of
vascular changes, we employed real time optical imaging. Mast
cell degranulation was confirmed histologically by examining mast
cells within tissue and by measuring histamine in venous flow
passing through the site of cold challenge.
As will be shown, the vascular response associated with mast cell
degranulation is rapid and extensive and is modified by the
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administration of an antihistamine, a proof of principle that the
effect of anti-allergic compounds that impact the consequences of
mast cell degranulation may be monitored using this unique
Confirmation of Mast Cell Degranulation
We first verified that our inducible procedures were accompa-
nied by histamine release and that such evidence of mast cell
activation correlated with the severity of the reactions. The mean
group time profiles for serum histamine for healthy controls and
patients with CUrt is shown in Figure 1A. Although there was
variability amongst patients (as indicated by SEM), histamine
peaked at 45.5 nM after 5 minutes of rewarming, as expected .
Histamine levels were then compared at baseline and 10 minutes
following cold hand immersion (CHI) at the peak of the vascular
response in order to evaluate the histamine peak related to severity
of the induced reaction. At 10 minutes, the mean histamine
concentration was significantly increased in patients (Figure 1B,
p=0.006), while that of controls remained unchanged (p=0.53).
Although there was expected variability amongst patients as
indicated by SEM, serum histamine increased the most in the
highest severity group after 10 minutes of rewarming (Figure 1C).
Furthermore, there was significant correlation between histamine
levels and disease severity (r=0.79, p=0.002, Figure S1) Thus,
our challenge procedure triggered histamine release reflecting
mast cell degranulation which related to the severity of the
Serum tryptase did not increase over 35 minutes, consistent with
previous reports  (Figure 1D, p=0.24, ANOVA). However,
compared to baseline, post challenge histological sections (n=4) of
dermis in a representative patient revealed increase in staining of
tryptase diffusely within dermal tissues (Figure 2, compare left
upper panel with right upper panel). Closer inspection of
individual mast cells revealed both increase of tryptase staining
in surrounding tissue and numerous extracellular mast cell
granules (compare left lower panel with right lower panel)
following cold challenge. Normal volunteer biopsies did not show
increased staining of granular tryptase post challenge. These
findings in association with the documentation of histamine release
provide evidence of mast cell degranulation following CHI in
affected subjects [14,15].
Real-time Imaging of Vascular Changes Associated with
Mast Cell Degranulation
Real-time images were collected in all subjects before and after
CHI using three cameras. Images of a representative CUrt subject
at baseline and upon rewarming 10 minutes after CHI are shown
in Figure 3. At baseline, blood flow (Figure 3A), temperature,
(Figure 3C), color index (Figure 3E), and gross macroscopic
changes (Figure 3G) are relatively homogeneous and of low
intensity across the dorsum of the hand. Ten minutes post-CHI, at
the peak of the vascular response dramatic changes are observed in
the regional temperature and vascular flow. As can be seen in
Figure 3B, superficial blood flow increased greatly in the hand
relative to the fingers and multiple distinct patches of high tissue
perfusion emerge. Similarly, in deeper tissue, IR detected increases
in temperature, implicating involvement of small vascular beds
that evolve to encompass nearby tissues but spare the digits
(Figure 3D). These changes were associated with an increase in red
blood cell concentration in superficial tissues (Figure 3F) which
clearly paralleled the visual changes in skin color and tissue edema
of both the hand and fingers (Figure 3H). Based on these
observations, it appears that small superficial vessels dilate in
response to cold challenge, thereby increasing superficial red cell
concentration, but the underlying larger arterioles constrict,
thereby reducing total blood flow in the fingers. For comparison,
images of a control subject, which show minimal changes following
CHI, are presented in supporting information (Figure S2). A real
time infrared temperature video of the vascular changes that occur
in a patient during CHI and the corresponding release of serum
histamine can be viewed from the link in supporting information
(Video S1). These results are consistent with but do not prove that
mast cell degranulation within cutaneous tissues is the one inducer
associated with alterations both in superficial blood flow and in
deeper small vascular beds.
We then analyzed the temporal sequence in changes in blood
flow associated with mast cell activation. Plotting the mean values
versus time of rewarming after CHI (Figure 4) revealed significant
differences in the temporal domain for blood flow (Figure 4A,
p=0.0006), temperature (Figure 4B, p=0.0017), and color
(Figure 4C, p=0.23). In affected patients, the increase in blood
flow was rapid, peaking at 7–9 minutes. Blood flow changes were
persistent and did not return to approximate baseline for 25
minutes. Control subjects where there was no mast cell activation,
had no change in blood flow (Figure 4A). Statistical analysis of
blood flow is shown in Figure 5A, by unpaired t-test with Welch’s
correction (p=0.004) and Figure 5B (maximal blood flow), by
rank-sum, p=0.003. Patients also displayed more rapid tissue
rewarming which somewhat exceeded baseline temperature at 15
through 30 minutes (Figures 4B, and 5D, maximum above
baseline). The tissue temperature in controls more slowly returned
to baseline (Figures 4B, 5E, time to maximum temperature). The
mean values for maximal color index were not statistically
significant between patients and controls (Figure 5G, p=0.53),
but the maximum was reached more slowly in those with CUrt
(Figure 4C; Figure 5H, p=0.018). The rate of recovery as
reflected in the time required to return to half of maximum
(Figures 5C, F and I) also revealed differences in subjects. Patients’
recovery was significantly slower than controls as assessed by blood
flow (Figure 5C, p,0.001) and color (Figure 5I, 19.5 min vs.
8.2 min, p=0.006), but not by temperature (Figure 5F, p=0.21).
This data is consistent with the conclusion that patients have an
exaggerated response to cold as evidenced by a significant increase
in blood flow which accelerates tissue rewarming and which is a
consequence of mast cell degranulation.
We next analyzed the apparent shunting of blood flow away
from the digits. A direct comparison of the spatial-temporal re-
warming profile of the hand and all fingers shows a modest
increase in deeper blood flow in a control (Figure 6A, C), whereas
there is a divergent pattern in a patient (Figure 6B, D) who displays
a rapid increase in hand but not finger temperature. Thus, blood
flow alterations that follow mast cell degranulation are associated
with pronounced disturbances in regional blood flow.
To further establish the relationship between mast cell
degranulation and vascular changes, we compared peak histamine
release in patients and healthy subjects to maximum rate of change
of imaging signals (Figure 7). The maximum rate of increase in
blood flow corresponds to the peak in time of maximal histamine
release (Figure 7A). For temperature and color index derivatives,
the peak at ,2.5 min post challenge corresponds to the sharp rise
of histamine levels in the patient group (Figure 7B, C).
Correlations were not detected in controls (Figure 7D, E, F). A
significant correlation was found between histamine levels and
blood flow and temperature change from baseline but not color
index (Figure S3). This analysis further confirms the association
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between histamine release as a surrogate marker of mast cell
activation and vascular changes in those with CUrt.
Antihistamines cause Decreased Dermal Blood Flow
Three patients were re-imaged during challenge testing while on
a standard regimen of cetirizine (10 mg daily) for a minimum of
one week. Blood flow signal decreased markedly in subject 1,
partially in subject 2 and was unchanged in subject 4 (Figure 8),
thus indicating that treatment with antihistamine restores normal
blood flow in some patients. These findings correlated with the
clinical response in each patient. The marked reduction in LSCI
blood flow signal in subject 1 is shown pictorially in Figure S4.
Thus, LSCI detected an objective reduction in superficial blood
flow response, which corresponded with symptom relief. These
data support the conclusion that documentation of changes in
vascular blood flow offers a way to begin to understand the
physiologic consequences of specific interventions on the vascular
changes associated with provocation of physical urticarias in
patients suffering from these disorders.
In this study we used real-time imaging technologies to better
understand vascular changes associated mast cell degranulation
within tissues. We found that the vascular response associated with
mast cell degranulation is characterized by increased blood flow,
thermal warming, vasodilation (Figure 3 and S2), and recruitment
of collateral circulation (Figure 6). Treatment with antihistamine
partially restores the normal pattern of superficial blood flow and
is associated with less tissue edema (Figure 8 and S4). To associate
these changes with mast cell activation, we documented a rise in
serum histamine following CHI in affected subjects (Figure 1A)
Histamine levels were elevated accordingly when patients were
stratified based on severity (Figure 1C, S1). Serum tryptase levels
remained unchanged (Figure 1D) although local release of tryptase
by mast cells was documented (Figure 2). While it is known that
tryptase is released by human mast cells together with histamine
in vitro , there are discrepancies between the detection of
these mediators in clinical disease [18,19] attributed to the fact
that mature tryptase occurs in complex with proteoglycan, which
Figure 1. Histamine data and severity scale. Time profile of histamine release (A) for healthy controls and CUrt patients. Panel B shows the
comparison of controls and CUrt patients at baseline and 10 minutes post-CHI for histamine. Panel C shows a comparison of normal control subjects
and patients for histamine levels stratified by severity scale at 10 minutes post-CHI. Significance level, **p,0.01. No elevation in tryptase was
detected in patients (D, p=0.42, RM ANOVA) or controls (p=0.48). In data not shown, no elevation in tryptase was detected through 120 minutes
following CHI. Data shown as mean 6 SEM. CHI challenge period is indicated with a horizontal bar in Panels A and D.
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limits diffusion into the vascular compartment while it can be seen
locally within the dermis associated with mast cells . The lack
of detection of serum tryptase despite a rise in histamine entertains
the possible contribution of histamine from basophils. This is
unlikely for the following reasons: First, tryptase is also found in
human basophils although the amount may vary considerably
. Thus the absence of tryptase in blood could also be used as
an argument against the involvement of blood born basophils in
the cold induced reaction. Second, Kaplan et al., reported in an
in vitro model of acquired cold urticaria that histamine was only
induced in chilled skin tissues and not released from chilled blood
leukocytes and purified basophils . Third, the serum of
patients with cold urticaria does not activate basophils . Thus,
these observations implicate mast cells as the primary source for
the histamine-dependent reaction in patients with CUrt.
LSCI, IR and PLC simultaneously acquired images during CHI
challenge. A pictorial comparison of the three modalities at
baseline and 10 minutes post-CHI (Figure 3) displays a clear
difference in dermal blood flow patterns and inflammatory
response in patients versus controls. In patients, the marked
inflammatory response is seen earlier in the hand than in the
fingers (compare Figure 3B, to Figure S4B). We were able to
characterize the onset, peak and rewarming of tissue in patients
and to identify hemodynamic markers. Using LSCI post-CHI,
patients displayed significantly higher maximal, more delayed
onset and slower recovery of mean blood flow (Figure 4A). IR
imaging detected a rapid onset and exaggerated temperature
response above baseline (Figure 4B). Color indexes using PLC
revealed differences in delayed time to reach maximal color and
recovery (Figure 4C). These findings support the conclusion that
patients with CUrt during cold exposure have an exaggerated
response to natural body heat preservation.
We demonstrated in a subset of individuals that the warming
pattern of the hand and fingers is homogeneous in comparison to a
patient with CUrt where there is a more rapid and dramatic
recovery of the palm versus the fingers (Figure 6). Given mast cell
density is similar in superficial and deep dermis , the cooling of
more tissue in the palm may lead to greater efflux of histamine and
other vasodilator mediators and thus greater blood flow. At the
other end of the spectrum, although the rewarming pattern
following cold exposure in those with Raynaud’s syndrome is
homogenous, it is much slower than controls due to dermal
episodic ischemia . Our observations provide experimental
support to those patients reporting variable regional responses to
When correlating microvascular changes to histamine, we
demonstrated that the highest rate of change in response correlates
with the rise in histamine (Figure 7 and Video S1). Repeat analysis
of three patients on cetirizine highlights the utility of imaging to
quantify a decreased superficial inflammatory response to cold
challenge following administration of an anti-allergic drug
(Figures 8 and S4). The spectrum of responses is likely a
consequence of the need to block other mast cell derived or
induced mediators in patients that have more severe pathology.
Figure 2. Tryptase-stained skin biopsy. Skin biopsy in CUrt patient stained for tryptase (red) at baseline at low (106, upper panels) and high
(606, lower panels) magnification and at 15 minutes following cold stimulation time test (CSTT-see methods).
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Using LSCI which assesses superficial blood flow, IR which
reflects changes in blood flow in superficial and subcutaneous
tissues and PLC which detects the skin reddening caused by both
moving and stagnant red cells in blood capillary loops close to
dermal papillae, we were able to examine the sequence of events in
a local inflammatory response that occurs at different vascular
plexuses within the skin microvascular bed. We thus observed that
the time of onset, max rate of change, and mean time to reach
maximum for three imaging signals did not dissociate (Figure 7).
This observation supports the conclusion that rewarming is
associated with vasodilator recruitment of microvasculature in all
vascular plexuses simultaneously. These vascular changes would
be expected to facilitate the inflammatory response by increasing
exposure of blood born cells and proinflammatory proteins to the
area of insult.
Inducible inflammation in cold urticaria is a unique model that
allows tissue mast cell dependent events to be studied in humans at
baseline and during challenge testing. We were able to thus make a
number of new and novel observations, which included the dermal
phenotype of microvascular hypereactivity, time to recovery and
Figure 3. Blood flow, temperature, and skin color images of a representative CUrt subject. Images at baseline (A, C, E, and G) and at 10
minutes post-CHI (B, D, F, and H) for a CUrt subject (Table S1, Subject 6). Panel A and B, show blood flow images by LSCI; C and D, the temperature
images by infrared (IR); E and F, the skin color images by polarized light colorimetry (PLC); and G and H, visible light photography. The blood flow
image in A has been scaled up by a factor of 4 for visibility.
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response to antihistamine. It is tempting to speculate that patients
with cold-induced urticaria are exhibiting an exaggerated protec-
tive response to cold whereby mast cell degranulation represents a
protective mechanism to maintain blood flow and thus prevent
more extensive tissue damage, much as has been suggested for
cutaneous vasodilatation provoked by pressure .
Materials and Methods
Patients with CUrt and non-atopic healthy volunteers between
6 months and 65 years of age provided informed written consent
under protocol 09-I-0126, approved by the Institutional Review
Board (IRB)/Ethics Committee of the National Institute of Allergy
and Infectious Diseases and adhered to Declaration of Helsinki
Principles. Informed written consent approved by the IRB was
completed by next of kin, legal caretakers or legal guardians on
behalf of minors/children. In addition, minors/children complet-
ed and signed a minor patient assent to participate.
Subjects and Demographics
The age range for patients (n=7) with CUrt was 12–46 years
(mean 28) (Table S1). The majority of patients were female (71%).
The average duration of symptoms was 11.4 years, with an
average severity of 9 out of a 15-point scale (Appendix S1) and an
average CSTT threshold of 3.7 minutes. The majority of the
patients were not dermatographic (14%), but were atopic (71%)
with a total average serum IgE of 189 IU/mL and a resting basal
average tryptase of 4.5 ng/mL. All patients had normal comple-
ment levels, negative cryoglobulins and undetectable cold agglu-
tinin titers. All subjects were negative for cholinergic, exercise
induced, pressure and local heat urticaria. The age range for
control group (n=6) was 12–56 years old (mean 39) (50% female).
On the initial evaluation and following a history and physical
exam, participants underwent standard challenge testing for CUrt
and the time for induction was established. Subjects were advised
to withhold antihistamines, leukotriene antagonists, and other
agents that could modify the induction of urticaria for 5 days prior
to testing. Patients returned within 3 months for challenge testing
with imaging and serial blood draws. Blood samples were stored
and analyzed for mediators. After withholding all medications for
7 days, three of seven CUrt patients representing the spectrum of
severity returned for additional challenge testing while on cetirizine
(10 mg/D) for a minimum of one week. Four patients fell within
the medium (6–10) and three within the high (11–15) severity
groups. All controls had a zero severity score.
Cold Stimulation Time Testing (CSTT)
To verify CUrt, all patients underwent CSTT using a 50 ml
glass beaker of ice water (0u–2uC) placed on the volar surface of
the forearm for 5 minutes, with observation of urticaria
development upon rewarming for 30 minutes. As a marker of
severity, the minimal time threshold to develop a singular
circumscribed hive was established (Table S1).
Imaging, Cold Hand Immersion (CHI), and Serial Blood
Prior to the challenge, an intravenous catheter was placed in the
basilic vein of the left arm. Patients were acclimated to room
temperature (22–24uC) and positioned for 15 minutes prior to
testing. While sitting upright, the palm of the left hand was placed
on a loosely strung tennis racket head adhered to a platform. The
racket grid maintained the hand above the table surface,
minimizing conductive heat exchange of the hand with the
platform and allowing even evaporation of residual water.
One minute baseline imaging was followed by CHI with the
hand submerged in cold water (10uC) to 2 inches above the wrist
for 5 minutes . After immersion, the hand was removed, dried
and returned to the same position on the grid, whereupon imaging
continued for 30 to 60 minutes. Serial blood draws were obtained
at baseline and 0, 2, 5, 10, 15, 20, 25, 35, 60, and 120 minutes
after immersion as shown in Figure S5.
Histamine and Tryptase Assay
Blood samples were drawn into serum separation tubes, with
serum aliquots stored at 280uC until analyzed. Serum histamine
was measured using a competitive enzyme immunoassay (SPI-Bio,
Bertin Pharma, France). Serum tryptase was measured by the
ImmunoCAP 100 System (Phadia Inc., USA).
Assessment of Vascular Response
Non-invasive optical imagers are able to measure microcircu-
latory events in the skin in real-time. These include laser speckle
contrast imaging (LSCI), infrared thermal imaging (IR), and
Figure 4. Imaging time profiles for healthy controls and CUrt subjects. Mean blow flow (A), temperature (B), and color index (C) are shown
for healthy controls (gray) and patient with Curt (black). For summary data, mean baseline was subtracted from each individual time profile, and then
the profiles were smoothed, down-sampled, and averaged based on subject groups. Significant differences between CUrt and control groups are
seen for LSCI (A) and IR (B) imagers, but not PLC (C) as calculated by 2-way ANOVA. The dotted horizontal line in panel B represents baseline
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polarized light colorimetry (PLC). Such imaging provides quan-
titative and qualitative measurements of skin blood flow circula-
tion, temperature and degrees of redness to correlate with clinical
manifestations of skin disorders. LSCI is sensitive to microcircu-
latory perfusion to 1.0 mm of skin depth and has been used to
evaluate perfusion dynamics in burns, wounds (laser Doppler)
, diabetics , and cigarette smokers . Skin temperature
measured with IR reflects dermal metabolism and inflammation,
Figure 5. Maximum response marker: differences between healthy control and CUrt groups for blood flow, temperature, and skin
color. Maximum value above baseline (A, D, and G) and the time it was reached (B, E, and H) were calculated for LSCI (A, B, C), IR (D, E, F), and PLC (G,
H, I). Recovery time marker: differences between control and CUrt groups for blood flow, temperature, and skin color. Time of recovery to reach half
of maximum for LSCI (G), IR (H), and PLC (I).
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providing a window into the physiologic status of the nervous and
vascular systems. IR detects dynamic blood flow and pyrogenesis
within 10 mm of skin depth and temperature gradients of
0.0156C. IR has been used to follow treatment responses to
melanoma,  assess facial temperature in non-verbal individuals
with severe motor impairment  and evaluate the delayed
rewarming process in Raynaud’s phenomenon . PLC is digital
color photography that objectively measures epidermal skin color
changes to 500 mm depth . Absorption of red light in tissue
remains relatively constant during challenge regardless of red cell
concentration; however, absorption of green light increases as red
cell concentration increases . The color index calculation is
proportional to the difference of red and green color bands,
divided by the red color band, the latter of which is a normalizing
factor to baseline skin color (i.e. melanin content). To interpret
color index, higher values occur when absorption of green light
increases, which corresponds with elevated relative red cell
LSCI, IR, and PLC cameras were positioned above the support
platform and focused on the dorsal surface of the left hand during
CUrt challenge testing.
The LSCI (Moor Instruments, UK) camera measures red blood
cell flux (velocity6concentration) in living tissue. The camera was
positioned 30 cm above the subject’s hand. Images were collected
at 5 Hz, and each image has 1136152 pixels/frame and covers
15 cm 620 cm. Required calibration was performed prior to the
The IR camera (Santa Barbara FocalPlane, Lockheed Martin,
USA) passively measures heat radiated from the skin within the
wavelength range of 3–5 mm. Images (6406512 pixels/frame)
were acquired at 2 Hz from a distance of 50 cm, covering an area
of 28 cm622 cm. To convert IR camera photon counts to
temperature units, a multiple point calibration was implemented
using blackbody standard (CI Systems SR-80, Israel) and a fifth-
order polynomial was applied to calibration curve within
temperature range between 28uC and 33uC.
The PLC device consists of a Canon 2Ti (Canon, Japan) camera
equipped with a Macro-Ring Lite flash that quantifies changes in
skin color, also visible to the eye. The flash and lens are both
polarized and are aligned to be cross-polarized relative to each
other. In this configuration, photons that reflect off the surface of
the skin are filtered. Photons that penetrate deeper into the skin
are depolarized and can pass through the lens filter. Images
(518463456 pixels/frame) were acquired every 15 seconds at a
distance of 35 cm, covering an area of 18 cm 6 12 cm. The
normalized difference between the red and green color bands was
used to quantify color . All three cameras were controlled
remotely by separate computers and acquired images were
displayed in real-time for visualization and saved for off-line
Figure 6. Comparison of the temperature recovery of a healthy control and CUrt subject. Region of interest (ROI) of the fingers and hand
of a healthy control (A) and a CUrt subject (B), and corresponding time profiles (C and D).
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Dermal Imaging Analysis
To remove motion artifacts, images of each modality were
aligned with a rigid-body transformation algorithm to a reference
image using the edges of the hand as fiducial points. To analyze
time profiles for blood flow, temperature and skin color, the same
region of interest (ROI) for each modality was drawn for each
subject, which included the area between the base of the wrist and
the knuckles. Next, the time profile of each modality was
calculated using the mean signal of the ROI of each image. In a
subset of patients, this ROI was compared to ROIs on the fingers
to follow the process of hand rewarming.
The mean of the one-minute baseline was subtracted from each
point of the time profile to normalize patients. An elliptical low-
pass smoothing filter (frequency cutoff of 0.01 Hz) was applied to
each profile. These time profiles were then down-sampled to one
point per 30 seconds for further analysis. Any residual motion
artifacts in the profiles were removed manually by interpolation
using a third-order polynomial. To investigate the rate of change,
the first derivative of each down-sampled time profile was
calculated as the difference between adjacent values divided by
the 30-second time interval. The derivative results in imaging units
per unit time. For example, AU/min is the rate of change in AU
per minute for LSCI.
Skin Tissue Immunohistochemistry
Skin biopsies were obtained at baseline and 15 minutes after
CSTT (5 min) on the challenge site, placed into 10% neutral
buffered formalin, embedded in paraffin and cut into 5-mm-thick
sections. Local anesthesia was performed just before biopsy and
did not delay the timing of the biopsy. Slides for immunohisto-
Figure 7. Association of histamine and imaging derivatives. Analysis of mean serum histamine levels for patients (A–C) and controls (D–F)
plotted against the composite derivative (i.e. rate of change) of imaging time profiles for all subjects (see methods) for blood flow (A, D), temperature
(B, E) and color index (C, F). For example, AU/min is a rate of change of AU with respect to time (dAU/dt). The data supports the association between
histamine release as a surrogate marker for mast cell degranulation and vascular changes in those with CUrt, but not healthy subjects.
Figure 8. Blood flow profile for CUrt subjects treated with antihistamine. Three CUrt patients were re-imaged using LSCI during CHI while
taking antihistamines (10 mg cetirizine). Blood flow time profiles for three patients (A, subject 1; B, subject 2; C, subject 4) before (black line) and after
(gray line) treatment. The region of interest used for this plot included only the area between the base of the wrist and the knuckles.
Mast Cell Dependent Vascular Responses
PLOS ONE | www.plosone.org9 February 2013 | Volume 8 | Issue 2 | e56773
chemistry were deparaffinized and stained using the Discovery XT
and Red Map Kit from Ventana Medical Systems (VMS, Tucson,
AZ, USA). The MAb to mast cell tryptase (aa1) (Abcam,
Cambridge, MA, USA) was diluted in Antibody Dilution Buffer
(VMS) 1:100 and incubated for 2 hours. A biotinylated undiluted
secondary antibody of Goat anti-Mouse SS Link, (Biogenex, San
Ramon, CA, USA) was incubated for 32 minutes, followed with
enzyme conjugate, and Fast Red chromogen.
Four parameters to characterize time profiles were calculated
for each imaging modality, including baseline, maximum, time to
maximum, and time to half-max post-max. Baseline was the mean
of one-minute imaging prior to CHI. Maximum and time to
maximum were the maximum signal after immersion and the time
to that point. The time to half-max was the time at which the
signal was equal to half of the maximum, (or for IR: equal to the
average of the minimum and maximum) and is an estimate of
recovery time. The correlation between clinical severity and
threshold values and these parameters was calculated using Prism
5 (GraphPad, USA).
Mean group time profiles for each subject group were
calculated. Two-way ANOVA analysis was conducted to test if
average response curves were significantly different. Data were
compared to a Gaussian distribution for verification that the data
was normally distributed using the Shapiro-Wilk Normality Test.
Data that were not normally distributed were analyzed using non-
parametric tests. Statistical results of p#0.05 were considered
significant. Error bars are 61 SEM.
Plot showing a significant correlation (p=0.002) between serum
Correlation of histamine with disease severity.
images of a representative healthy control subject.
Showing lack of response to cold challenge in comparison to
CUrt subjects (Figure 3). Images at baseline (A, C, E, and G) and
at 10 minutes post CHI (B, D, F, and H). A and B, blood flow
images by LSCI; C and D, temperature images by infrared (IR); E
and F, skin color images by polarized light colorimetry (PLC); and
G and H, visible light photography.
Blood flow, temperature, and skin color
sponse. Imaging values are compared to histamine levels collected
at 10 minutes post CHI for LSCI (A), IR (B) and PLC (C).
Significant correlation wasdetermined inblood flow (p=0.044) and
temperature (p=0.031) thus supporting the correlation between
mast cell degranulation and the vascular response.
Correlation of histamine with vascular re-
subject treated with antihistamine. LSCI images of subject
1 (Table S1) at baseline (A and C) and at 10 minutes (B and D)
post-challenge, untreated and treated with cetirizine 10 mg daily.
At baseline, no difference was observed in blood perfusion
between treated and untreated. At 10 minutes after CHI, a
decrease in blood perfusion was observed while on cetirizine.
Blood flow image of severely affected CUrt
sampling at defined time points that were collected
during imaging. Time sequence of blood draws, imaging, and
CHI test. Each procedure was performed during the time specified
by the shaded region. In some subjects, imaging was extended
from 30 minutes to a maximum of 60 minutes.
Challenge Timeline: Sequence of blood
graphics, severity rating, atopic status, baseline histamine, tryptase
and total IgE. Seven patients performed the CHI test. Prior to
CHI, a 15 point questionnaire was given to assess disease severity,
where 1 is low and 15 is high on the 1 to 15 range scale (Appendix
S1). Patients established a CSTT threshold time prior to CHI,
which was the minimum time of cold exposure that induced a
Patient Characteristics. Includes patient demo-
changes in sequence. Real time infrared imaging of patient
undergoing cold hand immersion shows rapid swelling and
increase in temperature above baseline. Sequential release of
histamine and corresponding IR profile of temperature sampled at
a region of interested between base of the wrist and knuckles. Rise
in histamine correlates temporally with maximal rate of change in
Histamine release and infrared temperature
naire completed by all subjects to determine urticaria severity.
Physical Urticaria severity Index. Question-
We acknowledge Chris Scully and Nitin Malik for their contribution to this
project. We thank Dr. Gert Nilsson for his technical advice with skin
polarized light colorimetry.
Obtained informed consent: MY SA. Conceived and designed the
experiments: JM AMG WML MY CN SA DDM HDK. Performed the
experiments: JM AMG WML NM MY CN AD DDM HDK. Analyzed
the data: JM AMG WML NM AD DDM HDK. Contributed reagents/
materials/analysis tools: JM AMG WML NM AD DDM HDK. Wrote the
paper: JM AMG WML NM MY CN SA AD DDM HDK.
1. Stone KD, Prussin C, Metcalfe DD (2010) IgE, mast cells, basophils, and
eosinophils. The Journal of allergy and clinical immunology 125: S73–80.
2. Pearlman DS (1999) Pathophysiology of the inflammatory response. The Journal
of allergy and clinical immunology 104: S132–137.
3. Barnes PJ (2011) Pathophysiology of allergic inflammation. Immunological
reviews 242: 31–50.
4. Galli SJ, Tsai M, Piliponsky AM (2008) The development of allergic
inflammation. Nature 454: 445–454.
5. KleinJan A, McEuen AR, Dijkstra MD, Buckley MG, Walls AF, et al. (2000)
Basophil and eosinophil accumulation and mast cell degranulation in the nasal
mucosa of patients with hay fever after local allergen provocation. The Journal
of allergy and clinical immunology 106: 677–686.
6. Sheffer AL, Tong AK, Murphy GF, Lewis RA, McFadden ER, Jr., et al. (1985)
Exercise-induced anaphylaxis: a serious form of physical allergy associated with
mast cell degranulation. The Journal of allergy and clinical immunology 75:
7. Miner PB, Jr. (1991) The role of the mast cell in clinical gastrointestinal disease
with special reference to systemic mastocytosis. The Journal of investigative
dermatology 96: 40S–43S; discussion 43S–44S, 60S–65S.
Mast Cell Dependent Vascular Responses
PLOS ONE | www.plosone.org10 February 2013 | Volume 8 | Issue 2 | e56773
8. Juhlin L, Shelley WB (1961) Role of mast cell and basophil in cold urticaria with Download full-text
associated systemic reactions. JAMA: the journal of the American Medical
Association 177: 371–377.
9. Murphy GF, Austen KF, Fonferko E, Sheffer AL (1987) Morphologically
distinctive forms of cutaneous mast cell degranulation induced by cold and
mechanical stimuli: an ultrastructural study. The Journal of allergy and clinical
immunology 80: 603–611.
10. Lawlor F, Kobza Black A, Breathnach AS, McKee P, Sarathchandra P, et al.
(1989) A timed study of the histopathology, direct immunofluorescence and
ultrastructural findings in idiopathic cold-contact urticaria over a 24-h period.
Clin Exp Dermatol 14: 416–420.
11. Cuss FM (1999) Beyond the histamine receptor: effect of antihistamines on mast
cells. Clinical and experimental allergy : journal of the British Society for Allergy
and Clinical Immunology 29 Suppl 3: 54–59.
12. Kaplan AP, Gray L, Shaff RE, Horakova Z, Beaven MA (1975) In vivo studies
of mediator release in cold urticaria and cholinergic urticaria. J Allergy Clin
Immunol 55: 394–402.
13. Schwartz LB, Yunginger JW, Miller J, Bokhari R, Dull D (1989) Time course of
appearance and disappearance of human mast cell tryptase in the circulation
after anaphylaxis. J Clin Invest 83: 1551–1555.
14. O’Mahony L, Akdis M, Akdis CA (2011) Regulation of the immune response
and inflammation by histamine and histamine receptors. The Journal of allergy
and clinical immunology 128: 1153–1162.
15. Haas N, Toppe E, Henz BM (1998) Microscopic morphology of different types
of urticaria. Archives of dermatology 134: 41–46.
16. Soter NA, Wasserman SI, Austen KF (1976) Cold urticaria: release into the
circulation of histamine and eosinophil chemotactic factor of anaphylaxis during
cold challenge. N Engl J Med 294: 687–690.
17. Grandel KE, Farr RS, Wanderer AA, Eisenstadt TC, Wasserman SI (1985)
Association of platelet-activating factor with primary acquired cold urticaria.
N Engl J Med 313: 405–409.
18. Lin RY, Schwartz LB, Curry A, Pesola GR, Knight RJ, et al. (2000) Histamine
and tryptase levels in patients with acute allergic reactions: An emergency
department-based study. J Allergy Clin Immunol 106: 65–71.
19. McClean SP, Arreaza EE, Lett-Brown MA, Grant JA (1989) Refractory
cholinergic urticaria successfully treated with ketotifen. The Journal of allergy
and clinical immunology 83: 738–741.
20. Cowen T, Trigg P, Eady RA (1979) Distribution of mast cells in human dermis:
development of a mapping technique. Br J Dermatol 100: 635–640.
21. Foster B, Schwartz LB, Devouassoux G, Metcalfe DD, Prussin C (2002)
Characterization of mast-cell tryptase-expressing peripheral blood cells as
basophils. The Journal of allergy and clinical immunology 109: 287–293.
22. Kaplan AP, Garofalo J, Sigler R, Hauber T (1981) Idiopathic cold urticaria:
in vitro demonstration of histamine release upon challenge of skin biopsies.
N Engl J Med 305: 1074–1077.
23. Gentinetta T, Pecaric-Petkovic T, Wan D, Falcone FH, Dahinden CA, et al.
(2011) Individual IL-3 priming is crucial for consistent in vitro activation of
donor basophils in patients with chronic urticaria. The Journal of allergy and
clinical immunology 128: 1227–1234 e1225.
24. Merla A, Di Donato L, Di Luzio S, Farina G, Pisarri S, et al. (2002) Infrared
functional imaging applied to Raynaud’s phenomenon. IEEE Eng Med Biol
Mag 21: 73–79.
25. Fromy B, Sigaudo-Roussel D, Gaubert-Dahan ML, Rousseau P, Abraham P, et
al. (2010) Aging-Associated Sensory Neuropathy Alters Pressure-Induced
Vasodilation in Humans. Journal of Investigative Dermatology 130: 849–855.
26. Wanderer AA, Hoffman HM (2004) The spectrum of acquired and familial cold-
induced urticaria/urticaria-like syndromes. Immunol Allergy Clin North Am 24:
27. Leutenegger M, Martin-Williams E, Harbi P, Thacher T, Raffoul W, et al.
(2011) Real-time full field laser Doppler imaging. Biomed Opt Express 2: 1470–
28. Kingwell BA, Formosa M, Muhlmann M, Bradley SJ, McConell GK (2003)
Type 2 diabetic individuals have impaired leg blood flow responses to exercise:
role of endothelium-dependent vasodilation. Diabetes Care 26: 899–904.
29. Pellaton C, Kubli S, Feihl F, Waeber B (2002) Blunted vasodilatory responses in
the cutaneous microcirculation of cigarette smokers. Am Heart J 144: 269–274.
30. Santa Cruz GA, Bertotti J, Marin J, Gonzalez SJ, Gossio S, et al. (2009)
Dynamic infrared imaging of cutaneous melanoma and normal skin in patients
treated with BNCT. Appl Radiat Isot 67: S54–58.
31. Nhan BR, Chau T (2009) Infrared thermal imaging as a physiological access
pathway: a study of the baseline characteristics of facial skin temperatures.
Physiol Meas 30: N23–35.
32. Taylor S, Westerhof W, Im S, Lim J (2006) Noninvasive techniques for the
evaluation of skin color. J Am Acad Dermatol 54: S282–290.
33. O’Doherty J, Henricson J, Anderson C, Leahy MJ, Nilsson GE, et al. (2007)
Sub-epidermal imaging using polarized light spectroscopy for assessment of skin
microcirculation. Skin research and technology : official journal of International
Society for Bioengineering and the Skin 13: 472–484.
Mast Cell Dependent Vascular Responses
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