polariscopic examination immunofluorescence

4
Figure 4-2 Elastic fibers. This Verhoeff-van Gieson stain
demonstrates the darkly staining normal elastic fibers of the skin.
Several special stains for elastic tissue are available.
The most commonly used stains are the Verhoeff-van Gieson stain (Fig. 4-2) or Weigert resorcin-fuchsin. Additional
techniques, such as the Luna stain and the Miller stain,
may allow better visualization of elastic fibers than traditional methods (4). These stains are beneficial in the diagnosis of anetoderma, connective tissue nevi, mid-dermal
elastolysis, and other alterations of elastic tissue.
The Giemsa stain is frequently used to highlight mast
cells. Giemsa contains methylene blue, a metachromatic
stain. The granules of a mast cell stain metachromatically
purple (Fig. 4-3).
POLARISCOPIC EXAMINATION
Polariscopic examination is the examination of histologic
sections under the microscope with polarized light, that is,
light from which all waves except those vibrating in one
plane are excluded.
■
LABORATORY METHODS
79
For polariscopic examination, two disks made of polarizing plastics are placed on the microscope. One disk is
placed below the condenser of the microscope and acts as
the polarizer. The second disk is placed in the eyepiece of the
microscope or on top of the glass slide and acts as the analyzer. When one of the two disks is rotated so that the path of
the light through the two disks is broken at a right angle, the
field is dark. However, when doubly refractile substances are
introduced between the two disks, they break the polarization and are visible as bright white bodies in the dark field.
Polariscopic examination is useful in evaluating lipid
deposits, certain foreign bodies, gout, and amyloid.
With regard to lipids, it is not fully known why
certain lipids are doubly refractile and others are not.
In general, cholesterol esters are doubly refractile, but
free cholesterol, phospholipids, and neutral fat are not.
Only formalin-fixed, frozen sections can be used for a
polariscopic examination for lipids.
Doubly refractile lipids are regularly present in the tuberous and plane xanthomas and xanthelasmata (but not always
in the eruptive xanthomas) of hyperlipoproteinemia, in the
cutaneous lesions of diffuse normolipemic plane xanthoma,
and in the vascular walls of angiokeratoma corporis diffusum
(Fabry disease) (see Chapter 33). Doubly refractile lipids are
present, as long as the cutaneous lesions contain a sufficient
amount of lipids, in histiocytosis X (Hand–Schüller–Christian
type) (see Chapter 26), in juvenile xanthogranuloma (see
Chapter 26), in erythema elevatum diutinum (extracellular cholesterosis) (see Chapter 8), and in dermatofibroma
(lipidized “histiocytoma”) (see Chapter 32).
Doubly refractile lipids are absent in lipid-containing
lesions, as a rule, in necrobiosis lipoidica (see Chapter 14),
in hyalinosis cutis et mucosae or lipoid proteinosis (see
Chapter 17), and in multicentric reticulohistiocytosis and
solitary reticulohistiocytic granuloma (see Chapter 26).
Among foreign bodies, silica causes granulomas
showing doubly refractile spicules. These granulomas are
caused either by particles of soil or glass (silicon dioxide)
or by talcum powder (magnesium silicate) (see Chapter
14). Wooden splinters, suture material, and starch granules are also doubly refractile. An example of polariscopic
examination is seen in Fig. 4-4.
Gouty tophi show double refraction of the urate
crystals if the crystals are sufficiently preserved. They are
preserved by the use of alcohol rather than formalin for
fixation (see Chapter 17).
Amyloid shows a characteristic green birefringence
in polarized light after staining with alkaline Congo red
(see Chapter 17).
IMMUNOFLUORESCENCE TESTING
Figure 4-3 Giemsa stain. Mast cell cytoplasmic granules are purple.
Elder9781451190373-ch004.indd 79
Immunofluorescence testing is a specialized technique
that is beneficial in the diagnosis of certain skin disorders
(5,6). Two immunofluorescence methods are commonly
08/08/14 2:05 am
80
LEVER’S HISTOPATHOLOGY OF THE SKIN
Table 4-2
Multistep Scheme for the Interpretation
of Cutaneous Direct Immunofluorescence
Figure 4-4 Polariscopic examination. In this talc granuloma,
polariscopy reveals hundreds of refractile foreign bodies within
the dermis.
used in dermatology: direct immunofluorescence testing
(DIF), which probes for immunoreactants localized in
patients’ skin or mucous membranes, and indirect immunofluorescence testing, which is used to identify and titer
circulating autoantibodies in the patient’s serum. A modified indirect immunofluorescence technique using the patient’s skin as a substrate, known as immunomapping, is
used to determine the site of cleavage or abnormalities in
the distribution of mutated proteins in various forms of
hereditary epidermolysis bullosa.
DIRECT IMMUNOFLUORESCENCE
Direct immunofluorescence testing has a valuable diagnostic role in several autoimmune and inflammatory mucocutaneous diseases, including autoimmune-mediated
blistering diseases, dermatitis herpetiformis, Henoch–
Schöenlein purpura (immunoglobulin [Ig] A vasculitis), and cutaneous lupus erythematosus. The role of
direct immunofluorescence as a diagnostic procedure
is important but not critical in other dermatoses, such
as dermatomyositis, cutaneous porphyrias, pseudoporphyria, lichen planus, and vasculitides other than
Henoch–Schöenlein purpura (7). Table 4-2 illustrates
a stepwise schematic for evaluation of immunofluorescence sections prior to making an immunopathologic
diagnosis.
Biopsy Techniques
A 3- to 4-mm punch biopsy is generally adequate. In the
group of autoimmune blistering diseases, an inflamed
but unblistered perilesional area is the ideal specimen.
Blistered-lesional sampling is the most common cause of
false-negative results. On the other hand, sampling too
Elder9781451190373-ch004.indd 80
Real vs. artifact
Relevant vs. irrelevant
Specific vs. nonspecific
↓
Location of immunofluorescence
(Epithelial, basement membrane zone, vascular)
↓
Dominant immunoreactant
(Immunoglobulin [Ig] G? IgA? IgM? C3? Fibrin?)
↓
Characteristics
(Granular? Linear)
↓
Diagnostic algorithm based on IF patterns
distant from the blistering also can cause false-negative
results. In a few cases of pemphigus, pemphigoid, and
epidermolysis bullosa acquisita, false-positive results can
occur in blistered lesions. Of note, due to the often focal
and skipping nature of the immunoreactants in dermatitis herpetiformis, a shave biopsy often provides a broader
surface with which to evaluate dermal papillae than a
punch biopsy.
The performance of an adequate perilesional biopsy
in mucosal lesions is often not feasible, and thus a high
incidence of false negatives and even false positives may
occur in these specimens. In patients with desquamative
gingivitis secondary to mucous membrane pemphigoid,
an easy way to obtain sampling is by the so-called peeling technique, in which rubbing the perilesional affected
gingivae with a cotton swab induces a “fresh peeling of
the mucosa.” In most cases of mucous membrane pemphigoid, the 180- and 230-kD hemidesmosomal antigens are
the autoantigens; therefore, the hemidesmosomes will be
available for interpretation in the peeled gingivae specimens, and a linear immunostaining with “capping” phenomenon is observed (8).
It has been reported that there is a theoretical higher
incidence of false-negative results in bullous pemphigoid
lesions from lower extremities; however, this finding has
not been confirmed by others.
In asymptomatic dermatitis herpetiformis patients
who have strictly adhered to a gluten-free diet for less
than 6 months, or even patients that have not done so but
remain lesion-free due to dapsone therapy, wide-shaved
specimens from the elbows or any other classically affected area will still show the typical IgA deposits at the
tips of dermal papillae (9).
08/08/14 2:05 am
4
In autoimmune and inflammatory disorders other
than autoimmune blistering diseases in which direct immunofluorescence plays an important role, the specimens
should be taken from lesional areas, including cutaneous
lupus erythematosus, dermatomyositis, vasculitides, lichen planus, cutaneous porphyria, and pseudoporphyria.
Transport and Processing of
Biopsy Specimens
Tissue for immunofluorescence studies should be obtained fresh and kept moist until it is quickly frozen. Skin
specimens can be kept on saline-moistened gauze and
transported immediately to the laboratory if it is nearby. If
it cannot be transported in less than 24 hours, the specimen should be put into Michel transport medium. This
medium is composed of 5% ammonium sulfate, the potassium inhibitor N-ethylmaleimide, and magnesium sulfate in citrate buffer (pH 7.25). This solution is stable at
room temperature but must be kept in a tightly capped
container to prevent absorption of CO2 and acidification.
Specimens stored in Michel medium are stable for at least
4 weeks at room temperature. Specimens stored in Michel
medium and kept in the refrigerator can be preserved for
several weeks or even months. This method of transportation has made the direct immunofluorescence technique
much more readily applicable. When the specimen is received in a laboratory, the ammonium sulfate is washed
out and the specimen is oriented and embedded in OCT
(optimal cutting temperature) compound and then the
specimen is snap frozen. The tissue is then sectioned at
6 μm. The frozen sections are incubated with antihuman
antibodies to IgG, IgA, IgM, C3, C5b-9, and fibrinogen.
These antibodies are linked to a fluorescent label such as
fluorescein isothiocyanate (FITC) to allow visualization
using a fluorescence microscope (7).
■
LABORATORY METHODS
81
Some cases of subepidermal blistering disorders
with deposition of immune reactants may be difficult to
differentiate from one another. The typical example is
pemphigoid and epidermolysis bullosa acquisita, both
subepidermal blistering diseases with C3 and/or IgG
deposition on DIF (Fig. 4-5). In these cases, a technique
called salt-split direct immunofluorescence often circumvents this problem. Direct immunofluorescence salt-split
skin analysis can only be performed if the specimen sent
for DIF is not already blistered. This technique consists
of thawing the frozen specimen formerly used for routine
direct immunofluorescence and incubating it in 1 M NaCl
for 48 to 72 hours, allowing for separation of the epidermis from the dermis. Following the incubation in NaCl,
new sections of frozen skin are cut and incubated with
antibodies linked to FITC, similar to the standard DIF
testing. This salt cleaves the basement membrane zone
through the lamina lucida, leaving the hemidesmosomes
on the epidermal side and deeper-seated proteins such as
type VII collagen and epiligrin on the dermal side of an artificially induced blister. Therefore, in virtually all cases of
bullous pemphigoid, the linear IgG immunostaining will
be localized on the epidermal side (occasionally epidermal
and dermal) and in epidermolysis bullosa acquisita on the
dermal side.
Direct Immunofluorescence Interpretation
Autoimmune Blistering Diseases
Sensitivity of direct immunofluorescence with active autoimmune blistering disease should be close to 100%. If
it is not, it is likely due to technical reasons (10). In pemphigus vulgaris (PV) and pemphigus foliaceus (PF), the
IgG immunostaining on the epithelial cell surfaces can
be granular and/or linear, giving a characteristic “chicken
wire” pattern. C3 staining may also be detected, and rarely,
IgA. Nonspecific patchy granular staining along the basement membrane is not uncommon, especially in mucosal
lesions. In paraneoplastic pemphigus the IgG “chicken
wire” immunostaining tends to be linear, thick, and homogeneous throughout the epidermis and mucosal specimens, including those from bronchi with or without a
concomitant linear basement membrane staining (11).
Lichenoid mucosal and even cutaneous lesions in paraneoplastic pemphigus tend to show focal granular IgG and
other immunoreactants along the basement membrane
without the typical “chicken wire” pattern.
Elder9781451190373-ch004.indd 81
Figure 4-5 Direct immunofluorescence. Bullous pemphigoid.
Continuous linear C3 staining along the basement membrane zone.
08/08/14 2:05 am
82
LEVER’S HISTOPATHOLOGY OF THE SKIN
Cutaneous Lupus Erythematosus
Direct immunofluorescence has a significant value in
the evaluation of patients with active cutaneous connective tissue disease. The intensity of the deposits of immunoreactants along the basement membrane in these
patients correlates with the degree of interface/lichenoid
dermatitis/mucositis.
In discoid lupus erythematosus, granular immune
reactants (IgG, IgA, IgM, and C3) are present along the
dermal–epidermal junction. The most common immunoreactant visualized with direct immunofluorescence is
IgM; in systemic lupus erythematosus and in subacute
cutaneous lupus erythematosus it is IgG. Of note, most
patients with anti-Ro-positive subacute cutaneous lupus
erythematosus may have a characteristic granular IgGspeckling pattern along the basement membrane and
throughout the epidermis. The lupus band test, originally
described as positive when granular IgG is present along
the basement membrane zone in specimens from sunprotected nonlesional areas, is rapidly being abandoned
due to its unreliability and the availability of more reliable
methods for the early diagnosis and prediction of systemic
disease in lupus erythematosus (Chapter 10) (12,13).
In cutaneous lesions of dermatomyositis, dense,
granular C5b-9 (membrane attack complex) deposition
at the basement membrane zone and upper dermal vessels along with that of weaker C3, IgG, and IgM deposits
is a quite characteristic immunofluorescence pattern that
often helps to distinguish dermatomyositis from lupus
erythematosus spectrum, namely in acute phases of these
diseases (14,15) (Fig. 4-6).
Cutaneous Vasculitides
Direct immunofluorescence evaluation is a very important
diagnostic tool in the workup of cutaneous small vessel
vasculitis, especially Henoch–Schöenlein purpura. The
best immunofluorescence diagnostic yield in Henoch–
Schöenlein purpura is obtained from 1- to 2-day-old lesions. As lesions get older, the IgA deposits get degraded
and cleared. Because most patients have older lesions at
the time of the evaluation, a high index of suspicion is required and exhaustive search for scant granular IgA deposits in very superficial papillary dermis is mandatory before
ruling out Henoch–Schöenlein purpura.
Hypocomplementemic urticarial vasculitis is another
small-vessel vasculitis in which direct immunofluorescence plays a critical diagnostic role. In this type of cutaneous vasculitis, granular IgG and C3 deposits are seen
in and around small dermal vessels and along the basement membrane zone. The presence of basement membrane granular immunostaining among other clinical and
serologic findings in patients with hypocomplementemic
urticarial vasculitis has led some authors to believe that
this vasculitis is no more than a subset of systemic lupus
erythematosus (16).
Other Autoimmune and Inflammatory Skin Diseases
Lichen planus lesions, mainly the mucosal variant, are
characterized by typical yet not pathognomonic, linear
and shaggy fibrinogen deposits and patchy granular IgM
and C3 along the basement membrane.
The direct immunofluorescence findings in cutaneous porphyrias are indistinguishable from those seen in
pseudoporphyria. These findings are characterized by
thick and glassy linear IgG and IgA deposits in superficial dermal vessels in a “doughnut pattern” and along the
basement membrane. It is believed that immunoglobulins
become trapped and bound to glycoproteins in a thickened basement membrane zone and degenerated blood
vessels in this disorder.
INDIRECT IMMUNOFLUORESCENCE
Indirect immunofluorescence is a semiquantitative procedure in which immunolabeling is carried out to evaluate
the presence and titer of circulating antibodies or specifically to localize antigen in the skin.
Figure 4-6 Cutaneous Ro-positive cutaneous lupus erythematosus (idiopathic or drug induced) and dermatomyositis direct
immunofluorescence patterns. Left: Ro-positive cutaneous
lupus erythematosus (idiopathic or drug induced). Granular
IgG-speckling “dusting” pattern throughout the epidermis
(keratinocyte cytoplasmic) with mild deposits at the basement
membrane zone and an often in vivo antinuclear antibody pattern
highlighting keratinocyte nuclei. Right: Dermatomyositis. Dense
granular C5b-9 (membrane attack complex) deposits along the
basement membrane zone and upper dermal vessels.
Elder9781451190373-ch004.indd 82
Indirect Immunofluorescence in the
Evaluation of Circulating Antiepithelial
Antibodies
Blood is drawn into a tube without anticoagulant, and
the serum is serially diluted. Substrates most commonly
used are 6-μm frozen sections of monkey esophagus, human salt-split skin, and murine bladder. The substrate is
incubated with serum dilutions for 30 minutes at room
temperature and then washed; antibodies bound to the
08/08/14 2:06 am
4
substrates are detected by incubation with fluorescein
isothiocyanate-labeled goat antihuman IgG and/or IgA.
Monkey esophagus is probably the best substrate for
the evaluation of antiepithelial surface antibodies specifically for PV. PF has a high incidence of false-negative
results with this substrate, and normal human skin can
be used as a substrate if PF is suspected and there is a
negative result using monkey esophagus. Low titers of antiepithelial surface antibodies up to 1:80 or even higher
can also be seen in control sera (17). In PV and PF, antidesmoglein antibodies give a “chicken wire” staining pattern (Fig. 4-7). It may be more prominent on superficial
epithelial cells, whereas in paraneoplastic pemphigus the
antiplakin antibodies give a pattern that is consistently homogeneous throughout the epithelium and sometimes is
even associated with immunostaining along the basement
membrane zone.
Transitional epithelium is a plakin-rich substrate, and
thus murine bladder is a common substrate for the screening of circulating antiplakin antibodies in paraneoplastic
pemphigus (18). Exceptional cases of PV and PF and pemphigoid can have concomitant low-titer antidesmoplakin
antibodies.
Monkey esophagus is also a useful substrate in the
indirect immunofluorescence screening for subepidermal
autoimmune blistering disease. However, human salt-split
skin renders better definition of the subtypes of subepidermal blistering disorders. Disorders characterized by antibodies to hemidesmosomal proteins BP180 and BP230,
including those seen in bullous and gestational pemphigoid, some cases of mucous membrane pemphigoid, and
linear IgA bullous disease, are associated with a linear immunostaining on the epidermal side (roof) of the salt-split
human skin (Fig. 4-8).
On the other hand, patients with circulating antibodies reacting against type VII collagen and antiepiligrin
■
LABORATORY METHODS
83
Figure 4-8 Bullous pemphigoid: thin wavy linear IgG
deposition along the epidermal (roof) side of salt split basement
membrane zone.
(laminin 5), as is seen in epidermolysis bullosa acquisita
(Fig. 4-9) and antiepiligrin mucous membrane pemphigoid (Fig. 4-10), respectively, have circulating IgG autoantibodies that bind the dermal side of the salt-split human
skin. In bullous systemic lupus erythematosus, staining is
also seen on the floor of the salt-split skin, and nuclear
staining of keratinocytes may be present (Fig. 4-11).
More sensitive and specific assays for the evaluation
of circulating autoantibodies, including enzyme-linked
immunosorbent assay (ELISA) for antidesmoglein and
anti-BP180 antibodies, immunoblotting and immunoprecipitation for pemphigoid, epidermolysis bullosa acquisita, and antiepiligrin, and immunoprecipitation for
paraneoplastic pemphigus, have been recently incorporated into the diagnostic armamentarium of autoimmune
blistering diseases.
ELISA Testing for Immunobullous Disorders
ELISA has been developed to detect autoantibodies associated with specific immunobullous disorders. The ELISA
Figure 4-7 Pemphigus vulgaris. Indirect immunofluorescence
with monkey esophagus substrate. Anti-IgG staining of epithelium
showing a “chicken wire” pattern.
Elder9781451190373-ch004.indd 83
Figure 4-9 Epidermolysis bullosa acquisita: thick “ribbontype” linear IgG deposition along the dermal (floor) side of saltsplit basement membrane zone.
08/08/14 2:06 am
84
LEVER’S HISTOPATHOLOGY OF THE SKIN
Figure 4-10 Antilaminin 5 (laminin 332, epiligrin)
pemphigoid: thin linear IgG deposition along the dermal
(floor) side of salt-split basement membrane zone.
test applied to immunobullous disorders uses the autoantigen as the target and screens the patient’s sera for autoantibodies. ELISA testing for autoantibodies reactive against
desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) and bullous
pemphigoid antigen 180 (BP180) and bullous pemphigoid antigen 230 (BP230) have shown diagnostic utility
in a number of studies. In one study examining sera from
pemphigus patients, ELISA to detect IgG autoantibodies
against Dsg 1 and Dsg 3 was performed on 317 controls,
82 patients with PV and 25 with PF (19). The Dsg 3 ELISA
was positive in all 34 patients with untreated PV and the
Dsg 1 ELISA was positive in all 10 patients with untreated
PF. When patients undergoing treatment were included,
the sensitivities fell to 95% and 92%, respectively, but still
compared favorably to the sensitivity of indirect immunofluorescence which was 79% in PV and 84% in PF. All PF
sera were negative in the Dsg 3 ELISA and the specificity
of both assays was 98% or greater. The Dsg 1 and Dsg 3
ELISAs also provided reproducible data which allowed differentiation of PV from PF.
The sensitivity and specificity of a commercially available BP180-NC16a domain ELISA has been compared to
that of indirect immunofluorescence (IIF) testing in the
evaluation of bullous pemphigoid (BP) and pemphigoid
gestationis (PG) (20). ELISA was performed on serum
from 28 patients (24 BP, 4 PG) and 50 controls. IIF testing
was performed on serum from 27 patients and 98 controls.
ELISA for BP180-NC16a had a sensitivity of 93% and specificity of 96% (P < .001), while sensitivity was 74% and
specificity 96% (P < .001) for IIF. These results indicate
that ELISA has a higher sensitivity than IIF testing, but
similar specificity. Further evaluation of controls who had
IgG deposition on the dermal side of salt-split skin on DIF
testing showed specificity for the ELISA of 100% (all four
cases negative) and 80% for IIF testing (one of five positive). Overall, ELISA has greater sensitivity and specificity
for BP or PG than does IIF. Together, these studies and
others suggest that ELISA testing for autoantigens is more
sensitive than IIF and is likely to be used with greater frequency in the future, given that larger numbers of samples
can be analyzed in a relatively short time period.
Immunofluorescence for the Evaluation
of Site of Cleavage in Hereditary
Epidermolysis Bullosa
This technique offers a practical, yet useful diagnostic tool
in hereditary epidermolysis bullosa by revealing the site of
the defect in these mechanobullous disorders. Thus, this
technique classifies these disorders into epidermolytic,
functional, and dermolytic categories (Table 4-3).
In brief, this technique is performed as follows:
A freshly induced blister is obtained by twisting a
Table 4-3
Indirect Immunofluorescence for the
Evaluation of Site of Cleavage in Hereditary
Epidermolysis Bullosa (HEB)
Type of HEB
Figure 4-11 Bullous systemic lupus erythematosus: thick
“ribbon-type” linear IgG deposition along the dermal (floor)
side of salt-split basement membrane zone in combination with
strong in vivo antinuclear antibody pattern in keratinocytes and
dermal cells.
Elder9781451190373-ch004.indd 84
Epidermolytic
(simplex)
Junctional
Dermolytic
Anti-type IV
Collagen
Anti-BP180
Immunostaining Immunostaining
Floor
Floor
Roof
Roof
Floor
Roof
08/08/14 2:06 am