Double Immunostaining with CD1a and CD68 in the Phenotypic Characterization of Indeterminate Cell Histiocytosis
Indeterminate cell histiocytosis (ICH) is a rare disorder in which histiocytic cells proliferate, expressing markers of both X- and non-X histiocytosis. Nevertheless, it is not totally clear if both types of markers are co-expressed by the same cell in this disorder, or on the contrary, the histiocytosis is made of two phenotypically different types of cells. Some recent reports seem to indicate this latter option, since there is a non-homogeneous distribution of the cells in the dermis. The ones in the most superficial part of the biopsy would lose some of their markers when moving towards the bottom part of the dermis. In order to check if there is co-expression of CD1a and CD68 by the same cell, we performed an immunohistochemical study with double staining, in a case of ICH of a 74-year-old male, who presented multiple yellowish papules in chest, back and both arms. Their sizes varied between 1 and 3 mm.
One of the biopsies from one lesion of the back showed a dermal histiocytic infiltrate, which expressed S-100, CD1a, Factor XIIIa and CD68 in the common immunohistochemical study. Birbeck granules were not found in the ultrastructural study.
Our results with the double stain for CD1a and CD68 demonstrated that most of the histiocytes expressed either one marker or the other. Nevertheless, some of the histiocytes of the infiltrate co-expressed both markers. In all the cases, the cells with this combined phenotype were mononuclear. Although CD1a was mainly expressed by the cells at the top of the dermis, some cells of the deep dermis kept expressing this marker. The cells expressing both markers were mostly found in the top part of the dermis. The multinucleate cells expressed only CD68, but not CD1a.
A. Fernandez-Flores; J. A. Manjon 2; F. Manzarbeitia 3
From the Services of 1Anatomic Pathology and 2Dermatology, Hospital El Bierzo, Fuentesnuevas, Ponferrada, Spain, the 1Service of Cellular Pathology, Clinica Ponferrada, Ponferrada, Spain, and the 3Department of Anatomic Pathology, Fundación Jimenez Diaz,
Vyšlo v časopise:
Čes.-slov. Patol., 44, 2008, No. 2, p. 37-39
Histiocytóza z neurčených buněk (Indeterminate cell histiocytosis – ICH) je vzácná choroba, kdy proliferující histiocytární buňky exprimují znaky jak X-, tak non-X histiocytózy. Nicméně není zcela jasné, zda oba typy znaků jsou společně exprimovány jedním typem buněk, či naopak, histocytóza je tvořena dvěma fenotypicky rozličnými typy buněk. Některé novější práce se přiklání spíše ke druhé z uvedených eventualit, protože rozložení buněk v koriu je nehomogenní. Buňky v nejpovrchnějších vrstvách biopsie by ztrácely část svých znaků při svém pohybu do hlubších vrstev koria. Abychom zjistili, zda dochází ke koexpresi CD1 a CD68 jedním typem buněk, provedli jsme dvojité imunohistochemické barvení v případu ICH u 74letého muže s mnohočetnými žlutavými papulemi na hrudi, zádech a obou pažích. Velikost papul byla 1–3 mm. V biopsii jedné z lézí zad byl v koriu infiltrát z histiocytů, které v rutinní imunohistochemii exprimovaly S-100, CD1a, faktor XIIIa a CD68. Elektronová mikroskopie nezjistila přítomnost Birbeckových granulí.
Naše studie s dvojitým barvením CD1a a CD68 prokázala, že většina histiocytů exprimovala buď jeden nebo druhý znak. Nicméně, některé z histiocytů v infiltrátu exprimovaly společně oba znaky. Všechny buňky s tímto kombinovaným fenomenem byly jednojaderné. I když exprese CD1a byla charakteristická převážně pro buňky v povrchu koria, ojediněle se nacházela i u buněk v hlubokém koriu.
Buňky exprimující oba znaky se nacházely většinou v povrchových vrstvách. Mnohojaderné buňky exprimovaly pouze CD68, nikoli ale CD1a.
Klíčová slova: histiocytóza z neurčených buněk – Langerhansovy buňky – CD1a – CD68 – S-100
cell histiocytosis (ICH) is alleged to be made of histiocytes that express some
markers that are characteristic of Langerhans’ cell histiocytosis, as well as
markers which are typical of non-Langerhans’ cell histiocytosis.
when evaluating some of the reports in literature, in which immunohistochemical
studies have been performed on ICH, many clues seem to indicate that although
most of the cells express S-100 protein, there are two different populations of
cells: one which expresses CD1a, and the other one which expresses CD68. Some
reports have remarked how the CD1a-positive cells are mainly found in the top
parts of the dermis, while the CD68-positive cells are found in deeper areas of
the dermis (8). Nevertheless, there is no direct evidence, that the cells
expressing CD1a and the ones expressing CD68 are different. In order to test
the hypothesis that ICH is really made of two phenotypically different
populations of cells, we performed a double immunostain in a biopsy
of an ICH case. The antibodies for CD1a and CD68 were used on the same section.
Each antibody was revealed with a different chromogen, so it could be
tested if the expression of CD1a and CD68 was present in the same cells or in
Materials and methods
A 74-year-old man
came to the consultancy due to some lesions that he had noticed on the trunk
since 4 months ago. We performed two biopsies of two lesions from the back of
the patient. One of them was studied by conventional microscopy and the other
by electronic microscopy. The first one was fixed in formaldehyde and processed
under the routine protocol for biopsies and embedded in paraffin, and sections
for hematoxylin-eosin and Giemsa were obtained. Sections for
immunohistochemistry were also obtained using the Dako REAL EnVision detection
system, peroxidase/diaminonenzidine (DAB)+, rabbit/mouse, for CD1a
(DakoCytomation, monoclonal mouse anti-human, clone 010, code M351), Factor
XIIIa (Master Diagnostica S.L., Granada,
clone AC-1A1), CD68 (DakoCytomation, monoclonal mouse anti-human, clone PG-M1,
code N1576), and S-100 (DakoCytomation, rabbit anti-cow, code N1517).
study for double staining was also performed with CD1a and CD68. While the
immunostaining for CD1a was revealed with DAB (brown color), the one for CD68
was revealed with aminoethylcarbazole (red color).
second biopsy was fixed in glutaraldehyde, and sections were obtained and
studied under electronic microscope.
The examination of the
patient revealed multiple yellowish papules on the chest, the back and both
arms (Fig. 1). They measured between 1 and 3 mm.
The routine morphologic
study demonstrated a histiocytic dermal infiltrate, made of mononuclear,
as well as of multinuclear cells (Fig. 2), with focal and mild emperipolesis of
lymphocytes by multinucleated cells.
immunohistochemical study demonstrated that most of the cells expressed S-100
protein and (in a slightly smaller proportion) CD68 and factor XIIIa (in
both mononuclear and multinucleate histiocytes). CD1a was expressed by
mononuclear but not by polynuclear histiocytes. Although cells expressing CD1a
were more numerous in the superficial dermis, they were also common in the deep
double-staining method was used, the results showed that most of the
histiocytes of the infiltrate expressed either CD68 or CD1a. Nevertheless, some
histiocytes co-expressed both markers (Fig.4). In all cases, the cells with
this combined phenotype were mononuclear. Although CD1a was mainly expressed by
cells at the top of the dermis, some cells of the deep dermis kept expressing
this marker. The cells expressing both markers were mostly found in the top
part of the dermis.
study did not show any evidence of Birbeck granules.
histiocytosis (ICH) was first described in 1985 (10) referring to a disorder
made of cells that expressed both CD1a and S-100, and that lacked Birbeck
granules in the ultrastructural study. Nevertheless, since electronic
microscopy is not so commonly used nowadays in the diagnosis of this entity,
the name is usually used to refer to a histiocytic disorder in which cells
shared macrophage- as well as Langerhans’ cell-markers (1, 2, 8).
The disorder is
considered to be made of indeterminate cells. In the past, the latter were
supposed precursors of the epidermal/dermal dendritic cell system (4), but are
at present considered more as members of the cutaneous dendritic system, which
would be on their way to the regional lymph nodes (7, 8). They are especially
prominent in proliferating epidermis, as well as in mucous membranes (3).
hallmark of indeterminate histiocytosis has been alleged to be the coexistence
of features of both X- and non X-histiocytosis (8). Among the
non-X-histiocytosis markers, macrophage markers such as CD68 or HAM 56 have
been proved to be expressed by IHC cells (6). CD68 is traditionally
considered as “not expressed” by Langerhans’ cell (LC) histiocytosis, although
sometimes LC can express it (5). In this latter case, the intensity of the
expression is lower than the one which is shown by macrophages (5). Among
the X-histiocytosis-markers which are expressed by IHC cells, one can mention
CD1a and CD1c. S-100 protein (usually expressed by Langerhans’ cells) is also
mentioned sometimes as a crucial marker in the diagnosis of ICH (8).
Nevertheless, it can also be expressed by non-Langerhans’ cell histiocytosis
(5, 9), CD1a seems to be more specific; although it can occasionally be
expressed by non-Langerhans’ cell histiocytosis; in these latter disorders the
form of expression is weak and focal (5).
The presence of
emperipolesis, to a certain limit, is not incompatible with the diagnosis
of ICH (5). Although it has been admitted that if prominent inflammatory
infiltrate plus emperipolesis are seen, some of these lesions would be better
considered as sinus histiocytosis with massive lymphadenopathy (5), the latter
is usually CD1a negative.
In spite of the
importance of the expression of both types of markers by the cell of ICH in the
diagnosis of this entity, some clues in literature seemed to indicate
a sort of zonation phenomenon in the expression of immunological markers.
For instance, while S-100 and CD45 are expressed by the majority of the cells
in the lesions, CD68 and CD1a seemed to be expressed only by a percentage
of cells (6). Moreover, CD1 is mainly expressed by cells of the superficial
dermis while the infiltrate in the lower dermis is reactive for
a variety of macrophage markers (8). Some authors interpret this peculiar
distribution as a loss of histiocytosis markers, from the surface to the
deep parts of the dermis, which would be due to the unphysiological conditions
of the skin for cells which should be present in the lymph node (8).
Our report shows that
at least some of the cells co-express both markers. It also shows that the
histiocytes that do so, are mainly located in the top part of the dermis. The
current findings are not necessarily contradictory to previous reports which
suggest a change in the phenotype of the histiocytes from the top to the
bottom parts of the dermis. It just shows that, in case such change really
exists, there is at least an intermediate stage in which cells co-express X and
non-X histiocytic markers.
would like to thank Dr. Miguel Angel Martínez González from the University
Hospital Doce de Octubre (Madrid, Spain) for having performed the
ultrastructural study of one of the biopsies for us. We also want to thank Mrs
Trinidad Carrizosa (Fundacion Jimenez Diaz, Madrid, Spain), for having
performed the double-staining technique, and Mrs Manoli Sánchez Fernández
(Hospital El Bierzo) for the beautiful immunohistochemical stains.
Fernandez-Flores, MD, PhD
Celular, Clinica Ponferrada
(00 34) 987 42 37 32
(00 34) 987 42 91 02
1. Berti, E., Gianotti, R., Alessi, E.: Unusual cutaneous histiocytosis expressing an intermediate immunophenotype between Langerhans cells and dermal macrophages. Arch. Dermatol., 124: 1250–1253, 1988.
2. Kolde, G., Brocker, E.B.: Multiple skin tumors of indeterminate cells in an adult. J. Am. Acad. Dermatol., 15: 591-597, 1986.
3. Lessard, R.J., Wolff, K., Winkelmann, R.K.: The disappearance and regeneration of Langerhans cells following epidermal injury. J. Invest. Dermatol., 50: 171-179, 1968.
4. Murphy, G.F., Bahn, A.K., Harrist, T.J., Mihm, M,C. Jr.: In situ identification of T6-positive cells in normal human dermis by immunoelectron microscopy. Br. J. Dermatol., 108: 423–431, 1983.
5. Ratzinger, G., Burgdorf, W.H.C., Metze, D., Zelger, B.G., Zelger, B.: Indeterminate cell histiocytosis: fact or fiction? J. Cutan. Pathol., 32: 552-560, 2003.
6. Rodríguez-Jurado, R., Vidaurri-de la Cruz ,H., Durán-Mckinster, C., Ruíz-Maldonado, R.: Indeterminate cell histiocytosis: clinical and pathologic study in a pediatric patient. Arch. Pathol. Lab. Med., 127: 748–751, 2003.
7. Romani, G., Schuler, G.: The immunologic properties of epidermal Langerhans cell as a part of the dendritic cell system. Springers. Semin. Immunopathol., 13: 265–279, 1992.
8. Sidoroff, A., Zelger, B., Steiner, H., Smith, N.: Indeterminate cell histiocytosis- a clinicopathological entity with features of both X- and non-X histiocytosis. Br. J. Dermatol., 134: 525–532, 1996.
9. Tomaszewski, M.M., Lupton, G.P.: Unusual expression of S-100 protein in histiocytic neoplasms. J. Cutan. Pathol., 25:129-135, 1998.
10. Wood, G.S., Hu, C.H., Beckstead, J.H., Turner, R.R., Winkelmann, R.K.: The indeterminate cell proliferative disorder: report of a case manifesting as an unusual cutaneous histiocytosis. J. Dermatol. Surg. Oncol., 11: 1111–1119, 1985.