Formalin deposition as artifact in

Copyright © 2009 John Wiley & Sons A/S
J Cutan Pathol 2009
doi: 10.1111/j.1600-0560.2009.01492.x
John Wiley & Sons. Printed in Singapore
Formalin deposition as artifact in
biopsies from patients affected
by a new variant of endemic
pemphigus foliaceus in El Bagre,
Colombia, South America
Background: Most autoimmune diseases occur sporadically;
however, endemic pemphigus foliaceus (EPF) is present in specific
locales restricted to some geographic rural regions mostly in South
America, Central America and in Tunisia (Africa). Its geographic
restriction makes it an invaluable natural model for studying how the
environment, genetic background and host response contribute to the
development of autoimmunity. We described a new variant of EPF in El
Bagre, Colombia, (El Bagre-EPF). When we examined the skin biopsies
from 10 patients and controls from the endemic area, we detected in a
systematic manner several types of pigmentation, sometimes
intracellular, and sometimes in the extracellular matrix in most biopsies.
Aim: We aim to determine the nature of this pigment in these skin
Methods: We studied 10 patients and 10 controls matched by sex, age
and work activity living in the endemic area by routine hematoxylin
and eosin (H&E).
Results: We were unable to find any bacteriological or parasitic
organism. Specifically, we searched for several tropical disease agents as
possible causative agents of this pigment. Iron stains and melanin
pigment bleaching techniques failed to determine the etiology of this
pigment. We then tried the removal of formalin pigment using picric
acid. The pigment was removed after very strong treatment with
different acids including picric acid.
Conclusions: Formalin pigment shares many properties with
hemozoin. In this case, the authors recommend the use of neutral
buffered formalin to prevent the formation of formalin pigment
especially after long periods of fixation when taking biopsies under
extreme temperature and environmental humidity.
Abreu Velez AM, Howard MS, Restrepo-Isaza M, Smoller B. Formalin
deposition as artifact in biopsies from patients affected by a new variant
of endemic pemphigus foliaceus in El Bagre, Colombia, South America.
J Cutan Pathol 2009. © 2009 John Wiley & Sons A/S.
Ana Maria Abreu Velez1 ,
Michael S. Howard1 , Marcos
Restrepo-Isaza2 and Bruce
1 Georgia
Dermatopathology Associates,
Atlanta, GA, USA,
2 Colombian Institute of Tropical Medicine,
Institute of Health Science, CES University,
Sabaneta, Antioquia, Colombia, and
3 Department of Pathology, University
Arkansas Medical Center, Little Rock,
Bruce Smoller, Department of Pathology,
University Arkansas Medical Center, Little Rock,
4301 West Markham St, Suite 783, Little Rock,
AR 72205, USA
Tel.: +501 686 5170
Fax: +501 296 1184
e-mail: [email protected]
Accepted for publication November 13, 2009
Abreu Velez et al.
Autoimmune diseases can be either organ-specific,
such as insulin-dependent diabetes mellitus, or systemic, such as lupus, and their association with other
autoimmune diseases in the same individual is not
uncommon.1 Because of the chronic nature of this
group of diseases, their treatment results in a tremendous cost burden to the Federal government as well
as to the health care system, in addition to the serious
reduction in the quality of life of the affected individuals. One of the best models to study autoimmunity
is the endemic form of pemphigus foliaceus [endemic
pemphigus foliaceus EPF].2 – 13 EPF was described in
focus in several countries in South America, Central
America and in Tunisia (Africa).
We discovered a new variant of EPF in El
Bagre, Colombia, South America (El Bagre-EPF).
This variant of pemphigus resembles Senear–Usher
syndrome (SUS), also known as pemphigus
erythematosus (PE).14 – 16 PE is defined as an overlap
of lupus and pemphigus, with the presence of
concomitant antibodies to the basement membrane
zone (BMZ) of the skin, as well as with deposition
of immunoglobulin and complement concomitant
with Intercellular stain between the keratinocytes
(ICS). Non-endemic pemphigus foliaceus (PF), or
pemphigus vulgaris (PV) lacks BMZ staining. El
Bagre-EPF predominantly affects seborrheic areas
and also the axilla of patients. Although both
EPF variants are endemic, fogo selvagem (FS) is
present in some areas of Brazil, affecting both
sexes equally, and shows highest incidence of onset
at 10–30 years of age.2 – 12 El Bagre-EPF cases
resemble SUS because of the presence of lupuslike lesions, with photo-exposed areas of the skin
being affected, and the presence of a lupus band
detected by direct immunofluorescence (DIF).14 – 16
El Bagre-EPF occurs in a gold-mining municipality
in El Bagre, Colombia; completely spares patients in
the first and second decades, affects predominantly
males of age 40–60 years and a few post-menopausal
females.14 – 16 El Bagre-EPF patients are exposed
to high levels of mercuric selenides, that act as
environmental pollutants, as well to several tropical
diseases.14,16 Sun exposure has been shown as the
most important exacerbating factor, along with high
temperatures and humidity. Figure 1 shows two
clinical pictures of a common presentation of this
disease and one typical immunofluorescence of the
skin of a patient affected by this disease.
We examined 10 cases of El Bagre-EPF. All
patients selected had the clinical features, lived
in the endemic area and showed cell surface
staining between keratinocytes by either DIF or
indirect immunofluorescence (IIF).9 – 11,14 – 16 The
clinical features of patients selected for study
included the presence of scaly, crusted erosions
on an erythematosus base confined mainly to socalled seborrhoic areas (e.g. face, scalp, upper part
of the trunk). Most commonly, the blisters were
superficial; however, these flaccid bullae are difficult
to find because they are transient and transform
into erosions. The erosions can become numerous,
showing a tendency to generalize (Fig. 1).16
The patient sera had antibodies to human IgG4
or total IgG against keratynocyte cell junctions in
glabrous skin.9 – 11 Other inclusion criteria were
that the patient serum immunoprecipitated a ConA affinity purified bovine tryptic 45-kDa fragment
of PF antigen or that the serum was positive by
immunoblotting for reactivity against desmoglein 1
(Dsg1), as well as for plakin molecules as previously
described.14 – 19
Skin biopsies
Biopsies from 10 randomly chosen patients and 10
controls matched for sex, age and work activity
were studied. Following local anesthesia without
epinephrine, lesional skin biopsies were from affected
areas on the chest, arms or face. Biopsies were
fixed in 10% buffered formalin and processed for
hematoxylin and eosin (H&E) staining. Normal skin
from the chest was used for the control group.
Iron staining
In order to evaluate the presence of the heme groups
secondary to malaria, we tested for iron in the skin.
We performed routine Prussian blue staining.20
Melanin bleach
We also considered that the observed pigment
could be melanin pigment. Therefore, we performed
melanin bleaching by using two identical sections
of each specimen, one was bleached and the other
was not. We used potassium permanganate 0.15 g,
50.0 ml of 0.3% sulfuric acid and 1% oxalic acid for
this procedure as previously described.20
Removal of formalin pigment
We examined the sections for the possibility of
the presence of ‘formalin pigment’ by dewaxing
of sections, rinsing in 100% alcohol, rinsing in
70% alcohol, rinsing in distilled water treating with
saturated alcoholic picric acid for 30 min–2 h and
washing in running tap water. If yellow staining of the
section persisted, we further rinsed in dilute lithium
carbonate, and finally rinsed in tap water again.20
Formalin stains in endemic pemphigus
Fig. 1. Shows a map of Colombia, with the El Bagre area including some pictures of the El Bagre municipality (urban area in a port). Most
patients with El Bagre-EPF lived in the rural area. Finally in the lower lane are shown some clinical features of the El Bagre-EPF patients and
one typical direct immunofluorescence using anti-human IgG conjugated with fluorescein isothiocyanate (FITC) showing intercellular stain
among the keratinocytes (green arrow), base membrane zone staining (blue arrow) and the black arrow shows a positive acrosiringium also
detected by these antibodies.
On routine H&E sections, several bizarre, pigmented
figures were seen (Fig. 2).
Neither the iron stain nor the melanin bleach
identified the pigment (Fig. 2). However, the formalin
destaining technique abrogated this pigment from the
Table 1 shows a summary of the different
characteristics of formalin pigment compared to
malaria pigment, with lipofuscin and other parasitic
and tropical diseases that prevail in El Bagre and
surrounding areas. The most common parasitic
diseases include malaria and leishmaniosis. In
addition to these, the gastrointestinal parasites
including Dirofilaria immitis (mostly in dogs) are also to
be considered.
We considered several etiologies for this strange
pigmentation seen in almost all biopsies from the
patients and controls from the endemic area. Our
first consideration was malaria because the El BagreEPF is predominant in a tropical area with all types
of tropical diseases prevalent all year-round.
Malaria pigment
Despite the worldwide public health impact of
malaria, neither the mechanism by which the
Plasmodium parasite detoxifies and sequesters heme,
nor the action of current antimalarial drugs is
well understood.21 – 23 The intraerythrocytic malarial
parasite uses hemoglobin as a major nutrient source.
Digestion of hemoglobin releases heme that the
parasite converts into an insoluble microcrystalline
material called hemozoin or malaria pigment. Some
authors purified hemozoin from the causative agent
of malaria in humans, Plasmodium falciparum, and used
infrared spectroscopy, x-ray absorption spectroscopy
and chemical synthesis to determine its structure.
The molecule consists of an unusual polymer of
hemes linked between the central ferric ion of
one heme and a carboxylate side-group oxygen
of another. The hemes are sequestered via this
linkage into an insoluble product, providing a
unique way for the malarial parasite to avoid
the toxicity associated with soluble heme.21,22 The
heme groups released from the digestion of the
hemoglobin of infected red blood cells are aggregated
into an insoluble material called hemozoin or
malaria pigment. Synthetic β-hematin (FeIIIprotoporphyrin-IX)2 is chemically, spectroscopically
and crystallographically identical to hemozoin.21 – 23
Some authors reported the crystal structure of
β-hematin determined using simulated annealing
techniques by analyzing powder diffraction data
obtained with synchrotron radiation.21 – 23 The
authors reported that the molecules were linked into
dimers through reciprocal iron–carboxylate bonds
to one of the propionic side chains of each porphyrin,
and the dimers form chains linked by hydrogen bonds
in the crystal.21 – 23
Abreu Velez et al.
Fig. 2. Shows different staining; some seem to be intracellular, some extracellular, some with bizarre forms and others resembling malaria
shistosomiasis. In the upper lane different microscopic pictures of the melanin bleach that unsuccessfully clear the pigment are shown. The
fourth lane of pictures shows the iron stain (blue) (red arrows) which does not colocalize with the formalin pigment.
We further considered dracunculiasis, onchocerciasis, schistosomiasis, trachoma and hydatidoses
based on the bizarre chitin-like structures that may
resemble some of these parasitic diseases.24 – 26 As
mentioned above, we also took into consideration
the diagnosis of schistosomiais as a possible causing
Commonly seen in
biopsies from
tissues containing
large hemorrhage
or tissues heavily
pigmented with
Shape and color
Formalin pigment
Malaria pigment
formed by the
breakdown of
hemoglobin of
RBC; usually
located in the
Kupffer cells
parasitized in the
Finely granular
pigment granules,
around the
Group of lipid
pigments residues
of lysosomal
digestion found in
cardiac, smooth
macrophage sweat
glands, liver,
kidney, adrenals,
nerve cells and
ganglion cells
Malaria pigment
It is brownish-black
pigment and
in polarized light
Lipofuscin or
May be present in
areas of old
hemorrhage or are
deposited in
tissues with iron
Melanins are pale
brown to dark
brown and even
black pigments
that are found
intracellular in the
melanins are
found in hair, skin,
retina and the
substantia nigra of
the brain
The shape of
Melanin pigment
Iron (or
Table 1. Summary of histopathologic characteristics of formalin pigment compared to malaria pigment and other pigmented structures
The heartworm is a
type of filaria, a
small thread-like
After infection, the
deposit under the
skin; then they
migrate to the
muscles of the
chest and
abdomen and into
the bloodstream to
the heart to reside
in the right
ventricle and in the
pulmonary artery.
Sometimes seen in
eye, brain, or an
artery in the leg
Dirofilaria immitis
Promastigotes that
reach the puncture
wound are
phagocytized by
macrophages and
transform into
multiply in infected
cells and affect
different tissues,
depending in part
on which
species is
involved. Usually
found on skin,
liver or spleen
promastigotes in
the sandfly vector
and as aflagellar
amastigotes within
Formalin stains in endemic pemphigus
Not available
100% alcohol,
rinsing in 70%
alcohol, then
0.15 g, 50.0 ml of
0.3% sulfuric acid,
and 1% oxalic acid
Formalin pigment
Special stains or
other annotations
Table 1. Continued
Strong oxidizing
agent such as
permanganate or
hydrogen peroxide
Sudan black B, long
acid fast and
Not available
for 5–10 min, and
with 0.1% nuclear
fast red for 5 min
Melanin pigment
Romanowsky, Fields,
Giemsa stains
Malaria pigment
Lipofuscin or
Visualization of the
Donovan bodies);
preparations of
peripheral blood or
aspirates from
marrow, spleen,
lymph nodes or
skin lesions
should be spread
on a slide and
stained with
Leishman’s or
Giemsa’s stain
Not available
Dirofilaria immitis
Acid phosphatase
Not available
Perl’s iron stain, the
Prussian blue
Pappenhein stain
Bleached by 10%
H2 O2
Iron (or
Abreu Velez et al.
Formalin stains in endemic pemphigus
agent of this pigment. We considered this entity,
based on the fact that we recently discovered several
autoantibodies to nerves and mechanoreceptors
on the skin of many El Bagre-EPF cases
(Abreu et al., submitted for publication). It is
known that schistosomiais is probably to be
responsible for a significant proportion of cases of
myelopathy occurring in endemic areas. Spinal cord
schistosomiasis is the most severe ectopic form of
schistosomal infection with the presence of lumbar
spinal cord infection in patients with transverse
myelitis, produced by Schistosoma japonicum. Several
of the El Bagre-EPF patients also describe lower limb
weakness associated with lumbar and/or lower limb
pain, urinary retention and intestinal dysfunction.
These symptoms are similar to those described when
spinal cord schistosomiasis is present.27 However, we
also considered that pigment was seen in biopsies
taken from the palms of the patients affected by
El Bagre-EPF. The cercaria could be introduced
through the skin, but it is extremely rare that the
schistosoma parasite could be located on the hands.
Usually, it is present in the vascular plexus of the
peritoneum and peri-vesical areas. The eggs can
pass through different organs, including the liver,
the intestine, and are discharged via urine or feces.
If the eggs are trapped in tissues, the formation of
granulomas is almost expected. Finally, El Bagre
and the surrounding area are not endemic for
schistosoma, which was the parasite most similar
in appearance to what we found in the biopsies.
Finally, we considered formalin pigment. Formalin
pigment may precipitate into the tissue, particularly
when the biopsies have been fixed in a formalincontaining fixative with an acid pH.20 Formalin
pigment artifacts are commonly seen in biopsies
from tissues containing abundant hemorrhage or
tissues heavily pigmented with blood.20 Other
authors reported encountering similar problems with
placental biopsies from patients from Tanzania
where the environmental temperatures and humidity
are also very high.28 The authors tested the accuracy
of standard and polarization microscopy in the
evaluation of 500 placental specimens from an
area of high malarial endemicity in Tanzania.28
They reported the use of polarization microscopy
to increase the sensitivity of detection of pigment
and parasites because of the marked birefringence
of hemozoin present in mature stage parasites which
accumulate in the placenta.28 The authors noticed
that in placental specimens with long periods of
fixation with formalin, formalin pigment formation
could be a problem, based on the fact that this
reagent shares many properties with hemozoin.
The authors recommended avoiding these problems
by using neutral buffered formalin to prevent the
formation of formalin pigment in placentas.28 Our
samples were fixed for longer periods and were
under extremely high temperature and humidity
in the jungle. This may be the problem, because
we used buffered formalin containers. Perhaps under
such extreme conditions the prevention of formalin
pigment formation maybe even more difficult.
This study brings to light a problem that many
researchers and clinicians may face when working
in extreme tropical conditions of temperature and
humidity. We highly recommend the use of fresh
formalin at pH 7, with the tissue fixed for a maximum
of 24 h to avoid these false positive artifacts that can
mislead the investigations, resulting in confusion with
parasitic and tropical diseases.
We acknowledge Drs Patricia Adem, MD, Wun-Ju Shie, MD,
PhD, MPH and Sherif R. Zaki, MD, PhD of the Centers for
Disease Control and prevention, National Center for Zoonotic,
Vector-Borne and Enteric Diseases, Division of Viral and Rickettsial
Diseases, Infectious Diseases Pathology Branch, for their excellent
second opinion on the biopsies. We also acknowledge Ms Kimberly
Hall at University Arkansas Medical Center and Mr Jonathan Jones,
for their excellent technical work. This work was performed with
funds from Georgia Dermatopathology Associates (GDA).
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