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research-article2017
VDIXXX10.1177/1040638717725783Charcot–Leyden crystals appear to be human-specificChoi et al.

Brief Communication

Journal of Veterinary Diagnostic Investigation

Charcot–Leyden crystals: do they 2017, Vol. 29(6) 904­–909

© 2017 The Author(s)
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DOI: 10.1177/1040638717725783
https://doi.org/10.1177/1040638717725783
jvdi.sagepub.com
report and literature review

Eunju Choi,1 Andrew D. Miller, Elizabeth Devenish, Makoto Asakawa,

Marina McConkey, Jeanine Peters-Kennedy

Abstract. The Charcot–Leyden crystal (CLC) is a major human eosinophil protein that readily crystallizes; these crystals
are common in eosinophilic diseases. Although anecdotal existence of these crystals is known in veterinary pathology,
definitive reports do not exist, to our knowledge. We identified eosinophilic crystals in a laryngeal myxosarcoma from a
2-y-old, spayed female, Labrador Retriever dog that were tentatively interpreted as CLCs. However, Ziehl–Neelsen acid-fast
stain was negative, arguing against CLCs. The crystals stained red with Masson trichrome, precluding collagen. Periodic acid–
Schiff and alcian blue were negative. The crystals stained positively with Okajima, and no myoglobin immunoreactivity was
detected, supporting their identity as hemoglobin crystals. In the absence of a hematologic abnormality, these crystals were
interpreted to be abnormal hemoglobin breakdown products. Protein sequence comparison was pursued to determine whether
a protein similar to CLC exists in mammals. Only 3 nonhuman primate species, the Sumatran orangutan (Pongo abelii), rhesus
macaque (Macaca mulatta), and cynomolgus monkey (Macaca fascicularis), had a sequence similarity of >80%. Of the
crystal-forming residues, 12 of 54 (22%) were different in the Sumatran orangutan and 15 of 54 (28%) were different in the
Macaca spp., which may affect the crystallization process. The lack of reports of CLCs in nonhuman species and our results
collectively suggest that CLCs are human-specific.

Key words: Charcot–Leyden crystal protein; histocytochemistry; sequence homology.

Charcot Leyden crystals (CLCs) are eosinophilic, refractile,
non-birefringent, hexagonal bipyramidal structures com-
monly encountered in human pathology. CLC protein is a
major constituent of the primary granules of eosinophils in
humans, comprising ~7–10% of total cellular protein.30 CLC
protein expression has been identified in human CD4+
CD25+ Foxp3+ regulatory T (Treg) cells.7 Initially, CLCs
were thought to be lysolecithin acylhydrolase, which has
lysophospholipase activity and interacts with the enzyme
lysophospholipase.31 However, lysolecithin acylhydrolase
has been renamed galectin-1017 based on the sequence simi-
larity to galectins and weak lactose-binding activity.8
In humans, CLCs are found in cytology or histology spec-
imens of eosinophilic inflammation caused by allergic dis-
eases such as asthma, parasitic infections, or any idiopathic
eosinophil-rich inflammation in any organ system.3,6 Tradi-
tionally, CLCs have been deemed to be a protein found only
in humans and nonhuman primates.9 In domestic species,
eosinophilic crystals present in cytology specimens with
eosinophils have been interpreted as CLCs.10 CLCs were also
previously believed to be the cause of eosinophilic crystalline
pneumonia in mice; however, it is now known that these crys-
tals are composed of a chitinase-like protein, Ym1.12 Given
the rarity of these crystals in research settings, recent publica-
tions have deemed these crystals nonexistent, at least in
mice.16,23 No work has been reported to prove or disprove the
presence of CLCs in veterinary species, to our knowledge.
We report herein in silico evidence that suggests that CLCs
are unique to humans plus a short review on eosinophilic
crystals that was triggered by the identification of eosino-
philic crystals in a laryngeal myxosarcoma of a young dog.
A 2-y-old, spayed female, Labrador Retriever dog was
admitted to the Cornell University Hospital for Animals
(Ithaca, NY) for surgical removal of a laryngeal mass. The
patient’s first clinical sign was a diminished bark that
appeared 10 mo prior. Over the subsequent months, the bark
was lost, and exercise intolerance developed during
walks. One month prior to the resection, a whistling or
wheezing sound was noted when exercising, which led to the
Section of Anatomic Pathology, Department of Biomedical Sciences
(Choi, Miller, Peters-Kennedy); Section of Small Animal Surgery,
Department of Clinical Sciences (Asakawa, McConkey); and Cornell
University, College of Veterinary Medicine, Ithaca, NY (Devenish).
1
Corresponding author: Eunju Choi, Section of Anatomic Pathology,
Anatomic Pathology Office, Department of Biomedical Sciences,
Animal Health Diagnostic Center, Cornell University, Ithaca, NY 14853.
aprilechoi@gmail.com
 Charcot–Leyden crystals appear to be human-specific 905

Figure 1.  Histologic features and staining properties of eosinophilic crystals within a canine myxosarcoma. A. The eosinophilic crystals
form radiating acicular spicules within the neoplasm. H&E. 200×. Inset: higher magnification of the eosinophilic crystals. H&E. 400×. B.
Using Okajima, the crystals (arrow) stained orange similar to erythrocytes (arrowhead). 400×. C. Using Masson trichrome stain, the crystals
were dark-red to purple. 400×. D. The crystals were negative using Ziehl–Neelsen acid-fast stain. 400×. E. The crystals were negative using
periodic acid–Schiff. 400×. F. Alcian blue staining did not highlight the crystals, but the matrix within the neoplasm stained blue. pH 2.5.
400×.

identification of a laryngeal mass on radiographs and via
laryngeal exam. On examination at Cornell, this finding was
corroborated on computed tomography (CT) scan, which
revealed a 1 × 1.3 × 1.9 cm laryngeal mass caudal to the
epiglottis with no evidence of lymph node metastasis.
At surgery, the mass was globoid, multinodular, smooth,
firm, tan-to-gray, and occupied ~85% of the airway. A single
stay suture was placed through the laryngeal mass, and the
mass was bluntly dissected until ~90% of the mass had been
removed. The resected mass was sent to the Cornell ana-
tomic pathology service. Five soft tissue fragments ranging
from 0.5 × 0.5 × 0.3 to 1.5 × 1.5 × 1 cm were received, fixed
in 10% buffered formalin for 24 h, processed routinely,
embedded in paraffin, and cut at 4-µm thickness. Histologic
sections were stained with hematoxylin and eosin (H&E),
Ziehl–Neelsen (ZN) acid-fast stain, periodic acid–Schiff
906
Choi et al.

Table 1.  Eosinophilic crystalline structures and their staining properties.

Histochemical stain and/or

Type Specific location or disease Composition immunohistochemistry Species
YM1 crystals Respiratory tract (lung), YM1 protein anti-Ym1 Mouse12
gallbladder, bile ducts, glandular
stomach, renal tubules
Hemoglobin crystals Variable Hemoglobin Okajima Rat,4,15 dog,5,11
horse,25 human2,33
Amianthoid fibers Palisaded myofibroblastoma and Collagen Masson trichrome Human,13,26 dog24
other sarcomas
Skeinoid fibers Neurogenic spindle cell tumor and Collagen Masson trichrome, periodic Human,21 horse29
gastrointestinal stromal tumor acid–Schiff (PAS)
Rosenthal fibers Gliosis and brain tumors, especially Intermediate anti-GFAP, Fluoro-Jade Human,32 dog,1,27
pilocytic astrocytomas filaments sheep14
Reinke crystals Leydig cell tumors Unknown Giemsa, Gram, Masson Human,19
trichrome, PAS, marmoset22
anti–3β-hydroxysteroid
dehydrogenase, anti-nestin
Charcot–Leyden crystals Eosinophilic infiltrate CLC protein Ziehl–Neelsen acid-fast stain Human6
and Masson trichrome

(PAS), alcian blue (pH 2.5), Masson trichrome, and Okajima
for hemoglobin and myoglobin. Immunohistochemistry for
myoglobin was performed (Leica BOND MAX IHC & ISH
Staining System, Leica Microsystems, Buffalo Grove, IL).
Formalin-fixed, paraffin-embedded sections were deparaf-
finized, rehydrated, and subsequently blocked with 3%
hydrogen peroxide (Leica). Antigen retrieval was performed
(Bond Epitope Retrieval Solution 2, Leica) for 30 min. Sec-
tions were incubated with anti-myoglobin (rabbit polyclonal;
Cell Marque, Rocklin, CA) for 60 min. Slides were then
incubated with a polymer (Bond Polymer Refine Detection
Kit, Leica) for 30 min. The slides were developed with DAB
(3,3’-diaminobenzidine) chromogen and counterstained with
hematoxylin. The external and internal positive controls and
negative controls were adequate.
Histologic examination revealed a multinodular, poorly
demarcated neoplasm expanding the submucosa of the lar-
ynx and effacing the normal architecture. The neoplasm was
composed of loose, haphazardly arrayed neoplastic spindle
cells forming streams and bundles on a background of pale
myxomatous matrix (Fig. 1A). Neoplastic cells had indis-
tinct cell borders, a small-to-moderate amount of eosino-
philic, elongated, fibrillar cytoplasm, and a round-to-elongated
nucleus with vesicular chromatin that lacked a prominent
nucleolus. Anisocytosis and anisokaryosis were moderate,
and 3 mitotic figures were present in ten 400× fields. Multi-
focally throughout the neoplasm, accounting for ~30% of the
sections examined, were areas of hemorrhage and necrosis
with small numbers of neutrophils and lymphocytes. Occa-
sionally embedded in the neoplasm were small deposits of
radiating eosinophilic crystalline spicules not associated
with hemorrhage or inflammation (Fig. 1A, inset).
The neoplasm was diagnosed as a myxosarcoma, and the
eosinophilic crystals were interpreted as Charcot–Leyden
crystals. However, unexpectedly, Okajima highlighted these
crystals orange as well as the erythrocytes in blood vessels, a
finding more compatible with hemoglobin or myoglobin
crystals (Fig. 1B). Immunohistochemical staining for myo-
globin was performed and did not immunoreact with the crys-
tals, ruling out myoglobin (data not shown). ZN acid-fast
stain has been shown to highlight CLCs in tissue sections,28
and Masson trichrome stains CLCs red-to-purple.3 Although
the trichrome did stain these crystals deep-red (Fig. 1C), pre-
cluding collagen, ZN was negative, suggesting that these are
unlikely to be CLCs (Fig. 1D). Alcian blue and PAS con-
firmed the mass to be a myxosarcoma based on the PAS- (Fig.
1E) and alcian blue– (Fig. 1F) positive myxomatous matrix
surrounding the neoplastic cells.
Eosinophilic crystals are rarely encountered in veterinary
histopathology with the exception of the mouse and rat. In
mice, buildup of Ym1, a chitinase-like protein, in the lung
causes eosinophilic crystalline pneumonia and can have sig-
nificant consequences.12 In the rat, hemoglobin crystals are
fairly common and are thought to be an intermediate break-
down product of hemoglobin.4,15 In our case, the eosinophilic,
refractile appearance of the crystals and unusual location
sparked investigation into these and other extracellular eosino-
philic deposits seen in histopathology. Our investigation
included reports of sclerotic collagen, amianthoid fibers, skein-
oid fibers, and Charcot–Leyden crystals from various species
(Table 1). Rosenthal fibers, seen in gliosis and in brain neo-
plasms such as pilocytic astrocytomas, and Reinke crystals
(seen in Leydig cell tumors of the testis) were excluded based
on the anatomic location. Amianthoid fibers are stellate-shaped
deposits of crystalline eosinophilic extracellular matrix com-
posed of collagen, most commonly encountered in the pali-
saded myofibroblastoma in humans.13,26 Skeinoid fibers are
eosinophilic stromal fibrillar aggregates seen in neurogenic
 Charcot–Leyden crystals appear to be human-specific 907

Figure 2.  Protein BLAST results using human Charcot–Leyden crystal (CLC) protein. A. Alignment tree comparing proteins identified
through protein BLAST. The dog, mouse, rhesus macaque, cynomolgus monkey, and Sumatran orangutan were chosen for comparison.
Note the distance between human and nonprimate species as well as the distance between these groups and mouse and dog. B. Sequence
alignment of human CLC and the 3 most similar species: rhesus macaque, cynomolgus monkey, and Sumatran orangutan. The amino acids
that are involved in crystallization are highlighted. Of the 54 amino acid residues involved in the crystal formation,18 12 (22%) amino acids
were different in the Sumatran orangutan and 15 (28%) amino acids were different in the rhesus macaque and cynomolgus monkey.

spindle cell tumor and gastrointestinal stromal tumor in
humans. Ultrastructurally, skeinoid fibers are short-spaced col-
lagen and appear as skeins of yarn.20 Amianthoid fibers, skein-
oid fibers, and sclerotic collagen are all composed of collagen
and stain blue with Masson trichrome. In our case, the crystals
were dark-red to purple when stained with Masson trichrome,
which suggested that the crystals were not composed of colla-
gen. In general, keratin, muscle fibers, cytoplasm, and fibrin
are red, and collagen, bone, and amyloid are blue with this
stain. PAS was applied for potential monoclonal immunoglob-
ulin deposition; however, the crystals in our case were PAS-
negative or a light-blue to purple and did not correspond to the
underlying matrix present throughout the neoplasm. Alcian
blue also confirmed the discordance between the matrix and
the crystals. The only positive stain that highlighted these crys-
tals was Okajima, a stain for hemoglobin and myoglobin. Neg-
ative immunoreactivity against myoglobin antibody ruled out
myoglobin.
Apart from the rat, it is believed that hemoglobin from
all other species crystallizes in vivo only if structurally
abnormal.5,15 In our case, no hematologic abnormality was
observed; however, a blood smear was not performed. Given
that these crystals were present within a tumor, the microen-
vironment of the neoplasm may have predisposed to hemo-
globin crystal formation. Interestingly, these crystals were
not associated with areas of hemorrhage, although hemor-
rhage was noted elsewhere in the neoplasm. The significance
of these crystals in this patient is unclear and was interpreted
to be tumor-related.
Although the neoplasm lacked eosinophils, the crystals
were initially interpreted as CLCs, and we speculated that
T-regulatory cells present in the neoplastic population could
have been the source. However, the histochemical staining
did not support the identification of CLCs. Furthermore, the
lack of reports of CLCs in the veterinary literature caused us
to believe that CLCs may be human-specific. In one study,
eosinophils isolated from humans and guinea pigs were
monitored to assess crystal formation after cell lysis using
light microscopy and electron microscopy.9 Crystal forma-
tion was present in human eosinophils but not in guinea pig
eosinophils, and this reaction to injury of the eosinophils of
humans and those of the guinea pig was concluded to be
characteristic for each of these 2 groups: primates, including
humans, and nonprimate mammalian species, including
guinea pigs.9
To further investigate the possibility of CLC formation in
nonhuman mammalian species, a protein BLAST search was
performed on mammalian protein sequences using default
settings of UniProt (http://www.uniprot.org). The query
sequence was human galectin-10 (UniProt entry Q05315
908
Choi et al.
v.3), and the target database was mammals. The Sumatran
orangutan (Pongo abelii) had a protein with the highest
sequence similarity at 87% with conservation of the domain.
The rhesus macaque (Macaca mulatta) and cynomolgus
monkey (syn. crab-eating macaque, long-tailed macaque;
Macaca fascicularis), common primates in research, had
~80% sequence similarity with conservation of the domain.
Of the 54 amino acid residues involved in the crystal forma-
tion of CLCs,18 42 amino acids were conserved in the Suma-
tran orangutan and 39 in the rhesus macaque and cynomolgus
monkey (Fig. 2). The differences in the amino acids involved
in crystal packing may explain the lack of crystal formation
in nonhuman mammalian species. Genome sequencing has
demonstrated that the CLC protein is absent in the mouse
genome, supporting the lack of reports of this structure in
mouse models of human disease in the literature.16 Similarly,
the lack of CLC protein expression in mouse eosinophils has
been described. 23
The presence of eosinophilic crystals in a myxosarcoma
in a dog led to research into CLCs in nonhuman mammalian
species. The crystals in the myxosarcoma were composed of
hemoglobin, and likely represent an intermediate product of
erythrocyte breakdown in the tumor microenvironment.
Although first considered CLCs, the lack of ZN staining,
positive Okajima staining, and negative immunoreactivity
against myoglobin antibody collectively support their iden-
tity as hemoglobin crystals. Simple in silico tools were able
to demonstrate that CLCs may be human-specific. Given
these results, caution should be used in interpreting eosino-
philic crystals in samples from veterinary species, and histo-
chemical stains should be used to support the identification
of these structures.
Acknowledgments
We thank the histopathology technicians at Cornell University Col-
lege of Veterinary Medicine for assistance with case processing.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The authors received no financial support for the research, author-
ship, and/or publication of this article.
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