Copyright © 2011 John Wiley & Sons A/S
J Cutan Pathol 2011: 38: 710–719
doi: 10.1111/j.1600-0560.2011.01746.x
John Wiley & Sons. Printed in Singapore
Journal of
Cutaneous Pathology
Autoantibodies to melanocytes
and characterization of melanophages
in patients affected by a new variant
of endemic pemphigus foliaceus
Background: Melanin and melanophages are commonly seen under
the basement membrane zone of the skin in patients affected by a new
variant of endemic pemphigus foliaceus in El Bagre, Colombia
(El Bagre-EPF).
Objective: Our study was conducted to determine the nature of these
pigmentary alterations.
Methods: We utilized clinical, histopathologic and immunologic
techniques including direct and indirect immunofluorescence,
immunohistochemistry, Bielschowsky staining and immunoelectron
microscopy studies.
Results: In the El Bagre-EPF patients, we detected dermal melanin in
melanophages and antigen-presenting cells, in close proximity to neural
and vascular markers. The melanophages consisted of a mixed
population expressing CD68, myeloid/histoid antigen and S-100
protein. By immunoelectron microscopy, the presence of
autoantibodies in proximity to melanin granules was confirmed within
the melanocytes utilizing 10-nm gold particles.
Conclusion: Dermal antigen-presenting cells, including
melanophages, seem to contain a diverse combination of molecules,
representative of an immunologic process where these cells are
engulfing both autoantigens and/or cellular debris in El Bagre-EPF.
Autoantibodies to discrete components of melanocytes were also
identified; the clinical and immunologic significance of these findings
remains unknown. Our work may provide a possible explanation of a
darkened complexion in patients affected by endemic pemphigus
foliaceus.
Keywords: autoimmunity, endemic pemphigus foliaceus, incontinent
pigment, melanocytes
Abreu Velez A M, Yi H, Googe PB Jr, Mihm MC Jr, Howard MS.
Autoantibodies to melanocytes and characterization of melanophages
in patients affected by a new variant of endemic pemphigus foliaceus.
J Cutan Pathol 2011; 38: 710–719. © 2011 John Wiley & Sons A/S.
We have previously described a new variant
of endemic pemphigus foliaceus in El Bagre,
Colombia, South America (El Bagre-EPF) that
resembles Senear-Usher syndrome. Senear-Usher
syndrome is considered an overlap disorder with
710
Ana Maria Abreu Velez1 ,
Hong Yi2 , Paul B. Googe Jr3 ,
Martin C. Mihm Jr4,5
and Michael S. Howard1
1 Georgia
Dermatopathology Associates,
Atlanta, GA, USA,
2
Robert P. Apkarian Integrated Electron
Microscopy Core, Emory University Medical
Center, Atlanta, GA, USA,
3 Knoxville Dermatopathology Laboratory,
Knoxville, TN, USA,
4 Departments of Dermatology and Pathology,
Harvard University Medical School, Boston,
MA, USA, and
5
Dermpath New England Laboratory, Boston,
MA, USA
Ana Maria Abreu Velez, MD, PhD,
Georgia Dermatopathology Associates, 1534
North Decatur Road, NE, Suite 206, Atlanta, GA
30307-1000, USA
Tel: +404 371 0077
Fax: +404 371 1900
e-mail: abreuvelez@yahoo.com
Accepted for publication May 23, 2011
features of lupus erythematosus, pemphigus foliaceus
and paraneoplastic pemphigus.1 – 3 Sinear-Usher
syndrome may occur in an endemic manner, with
systemic autoreactivity observed in approximately
one third of the patients; this autoreactivity is possibly
A new variant of endemic pemphigus foliaceus
because of autoreactivity against plakin molecules,
ubiquitously expressed in multiple organs.4 – 13
The conspicuous presence microscopically of
melanophages in the dermis of patients with endemic
pemphigus foliaceus, including the hyperpigmented
form of Brazilian fogo selvagem in Viera’s
original classification, remains a mystery.14,15
Several Brazilian authors have described significant
alterations in the skin color of the patients, which
some have referred to as a ‘change of race’;
specifically, these authors have reported a darkening
complexion.14 The presence of post-inflammatory
dyspigmentation and dermal melanophages is also
commonly noted as a consequence of various forms of
dermatitis. However, in diseases such fogo selvagem
and El Bagre-EPF, ultraviolet radiation seems to
exacerbate dyspigmentation; moreover, the clinical
lesions are more prominent in sun-exposed areas.1,3,5
Ultraviolet radiation is known to affect the entire
skin, including the basement membrane zone and
melanocytes.15
Based on these findings, we chose to further
investigate the nature of these pigmented dermal
cells utilizing tissue from patients and controls living
in the endemic area of El Bagre, as well as additional
controls with post-inflammatory pigmentary change
secondary to other forms of dermatitis.
Materials and methods
Subjects
A case-control study was performed utilizing patients
with Fitzpatrick phototypes (skin types) V and
VI, i.e. dark brown or black skin. We studied 30 patients who fulfilled the diagnosis of El
Bagre-EPF based upon clinical, epidemiological,
histopathological and immunological criteria; these
criteria were previously defined by us and others utilizing immunoblotting, immunoprecipitation,
ELISA, direct and indirect immunofluorescence and
immunohistochemistry.4 – 12 The skin biopsies were
taken from the patients’ lesional involvement and
were examined by hematoxylin and eosin staining
and by autometallographic assays.7 In addition, we
used the serum of the patients and controls for
indirect immunoelectron microscopy. We also tested
30 control patients from the endemic area; specifically their skin and sera, matched to the patients by
age, sex, work activity and place of living. One
exclusionary criteria for both cases and controls
was the stipulation that none were taking cloroquine or anti-malarial medicines to prevent inclusion of patients with drug-induced pigmentation.16
Five normal control sera from the United States
were used as negative controls. Non-endemic area
controls from other post-inflammatory pigmented
conditions were utilized, including one case each
of lichen planus, phytophotodermatitis, tinea versicolor (hyperpigmented subtype), fixed drug eruption
and bullous drug eruption.17 Finally, four cases of
inflamed dysplastic nevi were also used as controls.
We obtained informed consent from all patients. All
samples were tested anonymously to comply with
Institutional Review Board requirements.
Hematoxylin and eosin analysis
Staining was performed as routinely described.9 In
the El Bagre-EPF cases, the biopsies were taken from
active lesions most commonly involving the upper
chest. In the normal controls, biopsies were taken
from skin of the upper chest. Immunohistochemistry
was performed as previously described.9 – 14
Immunohistochemistry
We tested for reactivity to multiple immune
response antibodies, including monoclonal mouse
anti-human mast cell tryptase, Complement/C3,
Complement/C3c, Complement/C3d and polyclonal rabbit anti-human CD117(c-kit). For detection
of antigens within antigen-presenting cells, we utilized anti-human antibodies to HAM-56, CD68,
myeloid/histoid antigen (recognizing a human cytoplasmic antigen, i.e. L1-antigen or calprotectin),
CD1a, S-100, HMB-45, Mart-1/Melan A/CD63,
PNL2, tyrosinase and vimentin. For cell proliferation
analysis, we ultilized anti-human bromodeoxyuridine and anti-human topoisomerase II alpha (Dako,
Carpinteria, CA, USA). We performed immunohistochemistry utilizing a Dako dual endogenous
peroxidase blockage system with the addition of an
Envision dual link and following the manufacturer’s
instructions. We also tested for mouse anti-human
polyclonal von Willebrand factor and monoclonal
D2-40/podoplanin, to specifically identify vascular
and lymphatic endothelial cells, respectively. To
detect molecules of neural origin, we utilized (i) a
series of polyclonal rabbit antibodies, directed against
glial fibrillary acidic protein (GFAP) for neurofilaments, myelin basic protein and protein gene product
9.5 (PPG.9.5) and (ii) modified Bielschowsky staining (MBS). Table 1 summarizes the primary findings
among the El Bagre-EPF patients and controls from
the endemic area, as well from post-inflammatory
pigmentary change subjects.
Indirect immunoelectron microscopy
Post-embedding immunogold labeling was performed as previously described.15
Our MBS13,14 was performed as previously
described.
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712
Basically negative along
the BMZ in the
lesional areas
Basically negative along
the BMZ in the
lesional areas
Bromodeoxyuridine
Positive staining,
primarily along the
lateral sides of the
dermal papillary tips
in the inflammatory
infiltrate
Preserved in the middle
and deep dermis
Some cases exhibited
intraepidermal
positive staining,
while other cases
showed no staining
Decreased along the
BMZ in the areas of
the lesion
Lichen planus (N = 1)
Topoisomerase II
alpha
Myelin basic
protein
CD68
Myeloid/histoid
antigen
S-100
Stains
Linear staining along the
BMZ, and some
staining around
dermal blood vessels
under the
inflammatory process
Basically normal staining
pattern
Preserved in the middle
and deep dermis
Normal distribution, with
a few positive cells
around the upper
dermal blood vessels
A few positive cells noted
around the upper
dermal blood vessels
Positive in a few
inflammatory cells
around the upper
dermal blood vessels
Post-inflammatory
pigmentary changes,
excepting lichen planus
(N = 4)
Strong staining in the
epidermis, and within
dermal cells of the
nevi
Highlights neural tissue
in the middle and deep
dermis
Lost in central areas of
nevus cell nests, but
increased on sides of
these nests
Staining focally
decreased in the
nevus. Both nuclear
and cytoplasmic
staining overall
Very sparse along the
BMZ, but focally
expressed in the upper
layer of the epidermis
Sparse staining,
highlighting cells in
the dermal
inflammatory infiltrate
under the nevus
Dysplastic
nevus (N = 4)
Normal distribution
Normal distribution
Normal distribution
Positive staining along
the BMZ, and on
selected cells
surrounding upper
dermal blood vessels
Strong staining along the
BMZ, and in areas of
dermal inflammation
Normal distribution
Normal distribution
Normal distribution
Normal distribution
Normal distribution
Strongly positive
staining in the upper
papillary dermis, with
neural staining also
present in these areas
Focal staining seen
within melanophages
Normal distribution
Normal distribution
Strong staining in
blistering and dermal
inflammatory areas
Normal distribution
Normal distribution
Normal negative
controls/non-endemic
area (N = 5)
Decreased in the lesional
areas
El Bagre-EPF (N = 30)
Normal
controls/endemic
area (N = 30)
Table 1. Primary findings among the El Bagre-EPF patients and controls from the endemic area, as well from post-inflammatory pigmentary change subjects
Abreu Velez et al.
Mart-1/Melan
A/CD63
Tyrosinase
protein 1
Melanosome
(HMB-45)
Vimentin
Stains
Table 1. Continued
Almost lost in the BMZ
in active lesions
Relatively normal
staining pattern
In lesional skin, no
positivity is noted
along the BMZ;
positive staining noted
within papillary
dermal infiltrate
In the active lesions,
junctional zone
expression is
decreased
Lichen planus (N = 1)
Some strong expression
in lesional areas
Some strong expression
in lesional areas
Normal distribution
Normal distribution
Normal distribution
Normal distribution
Normal distribution
Accentuated staining at
the BMZ in lesional
areas
(i) Uniform staining of
epidermal
melanocytes in the
lesion. (ii) Nonuniform staining on
the dermal periphery
of the lesion
Strong expression on the
lesional areas
Focal expression in
lesional areas
Relatively normal
staining pattern
Relatively normal
staining pattern
Relatively normal
staining pattern
Normal distribution
In lesional skin, positivity
at the BMZ is present.
Focally positive
staining also noted in
the dermal infiltrate
Strong staining in nevus
cell nests along the
BMZ, especially along
sides of the dermal
papillary tips
Some staining around
dermal skin
appendageal
structures
Normal distribution
Normal distribution
El Bagre-EPF (N = 30)
Normal negative
controls/non-endemic
area (N = 5)
Normal
controls/endemic
area (N = 30)
Dysplastic
nevus (N = 4)
Post-inflammatory
pigmentary changes,
excepting lichen planus
(N = 4)
A new variant of endemic pemphigus foliaceus
713
Abreu Velez et al.
Statistical methods
Autoantibodies to mature melanocyte melanin granules were further statistically analyzed using Student’s
t-test to evaluate differences in morphology of the
reactive granules, and interpret our images. We considered a correlation to be present with a p value of
0.05 or less, assuming a normal distribution of the
samples
Results
Histopathology
Examination of conventional sections from the El
Bagre-EPF patients showed the focal presence of
melanophages in dermal papillae. The papillary
dermis showed a sparse superficial perivascular infiltrate of lymphocytes and histiocytes; neutrophils
and eosinophils were rare. At the level of the
superficial dermal vascular plexus, vessels were
dilated and/or surrounded by lymphocytes and
melanophages (Figs. 1 and 2).
Immunohistochemical studies
The differential intensity of staining of these antibodies between cases and controls was evaluated
qualitatively by the pathologist, as well as in
a semiquantitative manner by automated computer image analysis. Specifically, we utilized the
ScanScope CS system (Aperio Technologies, Vista,
CA, USA) with brightfield imaging at 20× and
40× magnifications. Tyrosinase antibodies showed
stronger staining in the cases compared with controls
from the endemic area, but showed no differences
vs. the control group of post-inflammatory disorders (Figs. 1–3). The presence of several antigenpresenting cells in the lower papillary dermis was
often seen, predominantly in the El Bagre-EPF cases
and in the control group with post-inflammatory
pigmentary changes. Some of the antigenpresenting cells were positive for the myeloid/
histoid antigen marker; this finding was noted in
90% of all the El Bagre-EPF cases, but not in the
control cases. The myeloid/histoid antigen-positivepresenting cells were seen in the papillary dermis and
around the upper dermal blood vessels. Other markers for antigen-presenting cells displayed similar positivity in the upper dermis, and included HAM-56,
CD68 and S-100. Of note, the positive markers in
antigen-presenting cell populations differed in acute
cases of pemphigus vs. chronic cases. In Table 1,
we summarize our main findings and differences vs.
the controls from the endemic area; however, the
El Bagre-EPF patient staining pattern was similar to
that in the group of post-inflammatory disorders.
714
We also found in the El Bagre-EPF cases that
several antigen-presenting cells contained materials
that were significantly positive for neural markers vs.
controls, including PPG.9.5 and MBP in addition to
melanin (p < 0.005). We used three standard deviations to assess significance, accounting for 99.7%
of the sample population being studied, assuming a
normal distribution.
Utilizing immunohistochemistry, we were able
to see that the basement membrane zone in the
El Bagre-EPF cases was usually labeled with antiComplement/C3 antibody; sometimes, the cytoplasm of epidermal keratinocytes, as well as cells
in areas around the papillary dermal blood vessels,
were also positive for anti-Complement/C3 and antiComplement/C3d. In addition, the mast cell tryptase
and CD117/c-kit markers were strongly overexpressed in the regions of the antigen-presenting cells,
especially in the patients (p < 0.005).
Of interest, when comparing the El Bagre-EPF
patients vs. the controls we also found that in many
of the controls with inactive post-inflammatory pigmentary lesions, the presence of melanophages was
decreased. In the controls with active lichen planus,
melanocytes were often decreased at the basement
membrane zone. In the El Bagre-EPF patients, we
detected our strongest staining with monoclonal
anti-bromodeoxyuridine; this marker highlights cells
which have incorporated bromodeoxyuridine into
their DNA during the S-phase of the cell cycle.
Indirect immunoelectron microscopy
At both 17 and 200 kV, we were able to see that
most El Bagre-EPF patients showed autoantibodies
to the basement membrane zone of the skin, precisely
in melanocyte cell processes in close proximity to
mature melanin granules. Please see Fig. 2, with red
arrows pointing to the 10-nm gold positive particles.
The melanocytes frequently rest on the dermal membrane, or bulge toward the dermis. We found two
types of melanin granules associated with our autoantibodies. The melanin granules were typed according
to their location in the melanocytes, and with respect
to their morphological characteristics which reflect
sequential stages in granule maturation. Specifically,
we distinguished (i) light melanin granules, in which
a structure resembling a fine network was apparent and (ii) dense melanin granules, which appeared
as uniformly dense masses surrounded by coarsely
granular, intensely osmiophilic shells. Perhaps most
interesting was the unexpected presence of autoantibodies in close proximity to the dense granules; these
autoantibodies were observed within the melanocyte
processes via 10-nm gold particles, and exclusively
seen in the El Bagre-EPF cases (p < 0.005) (Fig. 2).
A new variant of endemic pemphigus foliaceus
A
B
C
E
F
D
G
H
I
J
K
L
Fig. 1. A) Positive staining with S-100 in the papillary dermis; the red arrow highlights an antigen-presenting cell and the blue arrow points
toward basement membrane zone melanocytes. B) H&E staining showing numerous melanophages in the upper papillary dermis (brown
staining; blue arrow), C) PNL2 staining with strong, focal positivity at the basement membrane zone (brown staining; red arrow). D) Cells
showing strong staining for myeloid/histoid antigen in the dermis (brown staining; red arrows). E) A modified Bielschowsky stain confirms
that several of the antigen-presenting cells in the dermis contain neural antigens (brown staining; red arrows). F) Antigen-presenting cells
showing positive staining in the upper dermis with HAM-56 (brown staining; red arrow). G) Positive staining with tyrosinase at the basement
membrane zone (brown staining; red arrow). H) Positive CD1a staining in the epidermis (brown staining; red arrow), the blue arrow highlights
an antigen-presenting Langerhans cell in the dermis. I) Dermal antigen-presenting cells displaying positive staining with HAM-56 in the upper
dermis (brown staining; red arrow). J) PPG9.5 staining is shown by the red arrows, suggesting neural antigens exposed inside disease blisters.
K) Positive staining for Complement/C3 along the basement membrane zone (brown staining; blue arrow); the red arrow highlights grouped
dermal antigen-presenting cells. L) A typical pigmented plaque on the malar facial areas of a patient affected by El Bagre-EPF.
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Abreu Velez et al.
A
B
C
D
E
F
G
H
C
Fig. 2. A) Positive staining in dermal antigen-presenting cells with CD68 (brown staining; red arrow). B) Positive staining on an antigenpresenting cell with neurofilament antibody (brown staining; red arrow). The specific cell shown also stained positively for CD68 (not shown).
C) Positive staining of dermal antigen-presenting cells with S-100 (brown staining; red arrows). D) Positive staining of dermal antigen-presenting
cells with myeloid/histoid antigen antibody in a papillary dermal papilla (brown staining; blue arrow), and around the upper dermal vascular
plexus (red arrow). E) Strongly positive, focal staining for PNL2 along the basement membrane zone (brown staining; blue arrow). F) A
combination of bright field microscopy and a fluorescent filter shows some epidermal keratinocyte nuclei (light blue staining with Dapi). The
arrows show that deposited pigment (brown staining; red arrow) in the upper dermis is very close to the localization of desmoplakin 1 and 2
antibodies (red staining; blue arrow). G and H) Immunoelectron microscopy at 17 and 200 kV, respectively, showing positive staining of El
Bagre-EPF autoantibodies (grey tiny dot structures; red arrows).
Discussion
In this study, we present findings that contribute to an understanding of the clinically darkened complexion or change of race described in
patients affected by endemic pemphigus foliaceus.
Specifically, our findings highlight the future
scientific possibility of ultrastructurally confirming
disease autoantibodies within melanin granules. Our
data also suggest that antigen-presenting cells are
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actively processing diverse, amalgamated molecules;
these molecules likely include melanocytic, vascular and neural components. Significantly, the
basement membrane zone of the skin contains ultrastructural irregularities.18 The basement
membrane zone underlying the melanocytes contains
an electron dense plaque approximately 250–330 Å
in thickness.18 Melanocytic cytoplasmic filaments do
not seem to converge into this plaque. Instead,
a 50–70 Å cytoplasmic electron dense zone abuts
A new variant of endemic pemphigus foliaceus
A
B
C
D
E
F
G
H
I
Fig. 3. A) A dermal antigen-presenting cell (brown staining; blue arrow) in proximity to a lymphatic vessel (stained with D2-40; brown
staining; red arrow). B) Strong staining near dermal antigen-presenting cells with mast cell tryptase (brown staining; blue arrows). C) Strong
positive staining with Complement/C3c around upper dermal blood vessels, and inside the vessels (brown staining; blue arrows). D and E)
Positive staining on dermal antigen-presenting cells with Complement/C3d (brown staining; blue arrows). F and G) Positive staining of dermal
antigen-presenting cells with PPG9.5 (brown staining; red arrows). H) Positive staining on dermal antigen-presenting cells with myelin basic
protein, in areas where melanophages are present (brown staining; blue arrows). I) Strong reactivity for HAM-56 in the epidermis (brown
staining; red arrow), as well as in dermal areas where melanophages are present (brown staining; blue arrow).
the internal leaflet of the trilaminar melanocyte
plasma membrane and is directly apposed to adjacent
keratinocytes.18 Further, there are similarities and
differences between the hemidesmosomes abutting
keratinocytes and melanocytes, respectively. Finally,
the previously described basement membrane zone
cytoplasmic electron dense plaque is occasionally
interrupted with vesicles.18 Thus, we suggest that
small passages may be present within the basement
membrane zone, and these minute passages may be
relevant to El Bagre-EPF pathophysiology. Within
these passages, (i) thin, unmyelinated dermal nerves
could penetrate into the epidermis and (ii) immune
system autoantibodies and/or cell debris could pass
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Abreu Velez et al.
through the basement membrane zone. Notably, in
El Bagre-EPF and in many other post-inflammatory
disorders, melanophages are appreciated microscopically in focal areas of the dermis. Classically, the
content of these melanophages was believed to consist largely of melanin, which was present as a
result of passive fallout from the basement membrane zone following inflammatory disturbance. Our
study documents the presence of additional contents
within these cells, warranting further investigation
of melanophage pathophysiologic roles in various
dermatology diseases.
Similar to our previous reports, we were able
to detect autoantibodies and complement reactive
to the superficial nerves of the skin and to dermal
blood vessels in patients affected by El BagreEPF.14,15,19 However, our current data extend these
neural and vascular antigen findings into freely
mobile dermal cells, i.e. the melanophages and other
antigen-presenting cells. Further investigation of
these interrelationships is warranted.
A strongly significant difference was detected in
one data set between our El Bagre-EPF patient
data and that of the control group including both
(i) post-inflammatory disorders and/or (ii) diseases
with prominent melanin deposits. Specifically, the
antigen-presenting cells in the control group did
not show the same increase in staining with neural
markers observed with the El Bagre-EPF patients.
Classic studies in autoimmune blistering diseases
have investigated autoantibodies directed against
desmosomes in pemphigus and directed against the
basement membrane zone in bullous pemphigoid.
Our current study suggests the possibility of new frontiers in the investigation of cutaneous autoimmune
diseases. Specifically, we propose a higher order of
pathophysiologic complexity may exist and that further investigation may help to explain and address
clinical pigmentary alterations in EPF patients.20
Small non-myelinated nerves penetrate the epidermis; we suggest that the primary pathophysiologic
process could involve neural antigens in the disease process,14 in addition to antigens currently
documented in the medical literature. Alternatively,
neural antigen exposure could represent an epiphenomenon secondary to the primary pathophysiologic
process In addition, our immunoelectron microscopy
data suggests that the El Bagre-EPF autoantibodies
immunologically recognize unknown antigens inside
melanocyte cell processes, in close proximity to
mature melanin granules. Significantly, ultrastructural alterations within the melanosomes have been
previously described in selected cases of pemphigus
vulgaris.21 Further, disease blisters presenting within
melanocytic nevi have also been documented in
patients with pemphigus vulgaris.22,23
Further studies will be required to further
characterize the subtypes of dendritic and antigenpresenting cells24 in El Bagre-EPF, as well as those in
other cutaneous autoimmune blistering diseases and
in post-inflammatory pigmentary disorders. In addition, our findings of bromodeoxyuridine positivity
in the infiltrate associated with both El Bagre-EPF
and post-inflammatory dyspigmentation indicates a
need to characterize specific cell types observed in
association with these diseases.25
In summary, combining our current and past
findings,7,15 our observations indicate that El BagreEPF dermal antigen-presenting cells contain diverse
molecular components. These include (i) neural antigens, (ii) vascular antigens, (iii) mercuric selenides
or iodides and (iv) melanin. Secondly, the antigenpresenting cells are present in close proximity to
active disease inflammation, which is suggested by
our findings of deposition of complement, multiple immunoglobulins and multiple inflammatory
cells below the basement membrane zone. Finally,
our immunoelectron microscopy findings of disease
autoantibodies in close proximity to melanocyte
melanin granules warrants further investigation.
Specifically, these findings may contribute to an
understanding of the clinical darkening observed in
endemic pemphigus foliaceus patients.
Acknowledgements
We thank Jonathan S. Jones, HT (ASCP) at Georgia Dermatopathology Associates for excellent technical assistance.
Funding: Georgia Dermatopathology Associates, Atlanta, Georgia, USA (MSH, AMAV). The El Bagre-EPF samples were collected
from previous grants from the Embassy of Japan in Colombia,
DSSA, University of Antioquia and Mineros SA (AMAV), Medellin,
Colombia, South America.
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