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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_3099_Библиотеки_им_академика_М_И_Перельмана.pdf
Tissue Examination in a Diagnostic Procedure
77
Congo red dye is the most commonly used dye for the demonstration of
amyloid in tissues and remains the gold standard for the diagnosis. The dye
intercalates between beta-strands of the misfolded protein and gives a colour
between pink, red, and orange, sometimes called salmon pink colour. Several
modifications of Congo red staining were designed to increase specificity and
avoid false positive results; nevertheless the most reliable method seems to be
alkaline Congo red method [5, 6, 7].
Figure 3. Skin, Congo red, original magnification 200x. Multifocal amyloid deposits in
subepithelial interstitium.
Figure 4. Lung, Congo red (A) with polarizing microscopy (B) in original
magnification 200x, Sirius red in original magnification 40x (C). Deposition of
amyloid in vessel walls and in interalveolar septa.
Together with the Congo red positivity, the main diagnostic tool for
amyloid is the result of examination using polarizing microscopy. This method
works with a standard optical microscope and two specific filters, one is called
polariser and the second is analyser, which are placed between a light source
and the slide and between the slide and eyepieces respectively. Waves in the
Pavla Flodrova, Tomas Pika and Patrik Flodr
78
polarized light are in one plane and the change from regular to polarized light
occurs when the light passes through the filter. Using only one of the filters
does not change light in the field of view, but when planes of both filters are
perpendicular, the field of view is dark (sometimes called as “Cross-Nicol
position”). Congo red dye binds tightly to amyloid; molecules of the dye are
largely oriented in a linear and parallel manner on the amyloid fibrils [5].
When observed with perperdicular polarizer and analyser, the amyloid shows a
widely described phenomenon called green (or apple-green) birefringence.
When we look closely at this issue, a physical basis of this phenomenon is
much more complex and pathologists could often see more than one colour,
shades of yellow/green and blue/green, and sometimes yellow and blue colour.
Pure green colour can only be seen in ideal conditions, but more often there
are green and yellow or other colour combinations. Therefore, a more precise
definition was proposed, which says that Congo-red-stained amyloid,
examined between perpendicular polariser and analyser, shows anomalous
colours [8, 9].
However, Congo red-staining may also cause other difficulties in
evaluation. When the staining method is not optimal, the staining of collagen
or other commonly present tissue substances may be observed and any tissue
components, that bind Congo red, also exhibit adequate properties in polarized
light [5]. The result of the staining is also affected by the thickness of the
tissue section, with the most false negative cases in very thin sections.
Figure 5. Soft tissue, Congo red, original magnification 100x. In panel A Congo red
stained amyloid deposits viewed between perpendicular polariser and analyser in
polarizing microscopy showing green and yellow colour (B).
Tissue Examination in a Diagnostic Procedure
79
Figure 6. Thyroid gland, Congo red in original magnification 100x (A), Congo red in
original magnification 40x in polarizing microscopy (B). Large interstitial amyloid
deposits in the thyroid gland.
Therefore, it is strongly recommended, that the examination is performed
by an experienced pathologist.
BASIC COMPOSITION OF AN AMYLOID DEPOSIT
Amyloid deposits are composed of a main subtype-specific protein
component. A usually soluble monomeric protein is switched into pathological
fibrillar form with a high proportion of cross-β-sheet conformation and
stabilization via intra- and intermolecular interactions. This structure is highly
resistant to proteolytic degradation. Other supportive molecules known to be
associated with the amyloid deposits are namely proteoglycans,
glycosaminoglycans (heparan sulfate), an α1-antichymotrypsin, an
apolipoprotein E, an apolipoprotein A1, an apolipoprotein A4 and a serum
amyloid P component [3, 10, 11, 12,]. According to many authors, these
molecules are described as parts of deposits, however the relationship to
prognostic and predictive features of the disease or to therapeutic possibilities
has not been extensively investigated yet. The only well described substructure
of the amyloid deposits is an amyloid P component, resulting from the
nonfibrillar serum amyloid P component (SAP). SAP is a normal human
homopentameric plasma glycoprotein and belongs to the pentraxin protein
family together with the C-reactive protein, the acute-phase protein. A
characteristic property of this protein family is calcium-dependent ligand
binding. SAP binds avidly but reversibly to amyloid fibrils of all types and the
amyloid deposits become stabile and more resistant to proteolysis and
phagocytic cells due to this linkage [3]. Unlike the other abnormal
Pavla Flodrova, Tomas Pika and Patrik Flodr
80
proteinaceous materials, amyloid is not cleared from extracellular space by
physiological mechanisms, because no cellular or inflammatory reactions are
initiated [2]. According to the novel experimental works, SAP is supposedly
one of the most effective therapy targets using depletion of SAP from plasma
and application of an anti-SAP antibody, which trigger reactions caused by
macrophages resulting in the removal of the amyloid deposits [2, 13, 14].
Complex knowledge of the composition of amyloid led to a design of the
universal proteomic signature for amyloidosis, which may solve for example
Congo red-negative cases of subcutaneous fat aspiration samples. As a marker
– universal amyloid proteome - presence of at least two of the three proteins
was proposed – apolipoprotein E, serum amyloid P component, and
apolipoprotein A4. Presence of apolipoprotein A1 cannot be used as a marker
due to its regular presence in adipose tissue [11].
IMMUNOHISTOCHEMICAL EXAMINATION
Diagnostic confidence of the correct amyloid subtype is crucial to an
appropriate therapy selection. Problematic issue of simultaneously present
monoclonal gammopathy and hereditary amyloidosis in patients with systemic
form of amyloidosis has been already studied with the occurrence of this
phenomenon ranging from 3% to 10% [15].
Immunohistochemical (IHC) staining is a method used globally in the
diagnosis of tumours and it is widely available and easily applicable in most of
the pathology laboratories. An immunologic approach to the identification of
amyloid fibril constituents is usually preferred. The indirect IHC method is
based on binding of a primary antibody to a target antigen in the tissue. Both
polyclonal and monoclonal primary antibodies are routinely used; polyclonal
primary antibodies recognize several epitopes while monoclonal primary
antibodies are specific to one epitope only. A secondary antibody is
conjugated against the primary antibody with any type of reporter molecules
and this modification brings an amplification of the signal.
Although new diagnostic methods were proposed recently, the
immunohistochemical assessment of the amyloid type remains the essential
diagnostic approach even in big centres with modern technical equipment. The
advantages include the use of archival formalin-fixed paraffin embedded
(FFPE) material, global familiarity with the method, low-maintenance
equipment, and favourable price-benefit ratio. Amyloid deposits in tissue
samples are quite often found incidentally; therefore the only material for
Tissue Examination in a Diagnostic Procedure
81
further examination is the FFPE sample only. The FFPE sample can be
moreover easily sent to a highly specialised diagnostic centre to make a correct
diagnosis.
Figure 7. Myocardium, Sirius red in original magnification 100x (A),
immunohistochemical staining anti-transthyretin in original magnification 40x (B).
Amyloid forms plaques in the myocardial interstitium with positive
transthyretin staining.
Figure 8. Myocardium, immunohistochemistry, anti-kappa, original magnification
100x. Kappa-positive interstitial deposits of amyloid.
Antibodies are applied against fibrillar amyloidogenic proteins for
subtyping of amyloid deposits. A universal amyloid constituent part, as the
Serum amyloid P component, can be determined as a general
immunohistochemical amyloid marker.
Pavla Flodrova, Tomas Pika and Patrik Flodr
82
The role of immunohistochemistry has been already studied according to
its specificity and sensitivity. Some problematic issues are known in IHC
detection of amyloid antigens and these result in weak or lack of staining or
nonspecific staining. Most of these problems are connected with designation
of kappa and lambda light chains in AL amyloid, but they may also arise in
other proteins. Several moments play the key role, for example conformational
differences between native and fixed tissue, heterogeneity of variable domains
of light chains, conformational changes with light chain fragmentation, and
antigen masking during the process of amyloid fibril formation, genetic
mutations of fibrilar amyloidogenic proteins, and also contamination of
amyloid by serum proteins [15, 16, 17, 18, 19, 20]. Together with some
intrinsic natural features of light chains, the preanalytical phase of the tissue
processing can be a significant influencing factor, especially the way and
duration of the tissue fixation. Cases of false-positive staining may arise with
the use of an anti-transthyretin antibody and this pitfall may lead to
misdiagnosing hereditary and AL amyloidosis, especially in examination of
myocardial tissue in patients with heart amyloidosis [17, 19].
To avoid the abovementioned difficulties some centres apply more than
one antibody against kappa and lambda light chains in the standard diagnostic
process including non-commercially available antibodies [15]. Using of
unfixed tissue (frozen tissue) is believed to give better results [15, 16].
Bearing all that has been stated in mind, it is recommended to involve
multiple methods in a diagnostic process, especially in cases of uncertain or
non-specific outcomes of the IHC method, or of disagreement in clinical
presentation and IHC findings. Currently, a proteomic diagnostic approach is
usually used in these problematic cases. In some centres the proteomic
analysis is applied in any case of amyloidosis and it becomes the new gold
standard replacing the previously dominant immunohistochemistry [16].
FLUORESCENT EXAMINATION
Fluorescent examination of tissues is possible along with Congo red and
Thioflavin T, or as an immunologic method based on direct
immunofluorescence.
Congo red dye serves as a fluorochrome dye when bound between
amyloid fibrils. Congo red fluorescence (CRF) is supposed to be more
sensitive compared to a Congo red-stained bright field and polarized
microscopy and it can be easily performed and the results interpreted [21].
Tissue Examination in a Diagnostic Procedure
83
Figure 9. Kidney, Congo red in original magnification 100x (A) and
immunohistochemical staining anti-lambda in original magnification 200x (B).
Amyloid in glomeruli and vessel walls, deposits show Ig-lambda positivity.
Another fluorochrome dye is Thioflavin T and similarly to Congo red
staining the procedure can be performed on FFPE tissues. The staining
procedure is not difficult and it detects very small deposits of amyloid, but it
brings the risk of non-specific staining caused by binding on other structures in
the tissue – keratin, nucleic acids, and elastin fibrils, etc. [22, 23, 24].
Standard immunofluorescence is usually performed in renal biopsy
samples. In some institutions also the other types of tissues are examined this
way under the standard investigative procedure, for example tissue samples
obtained by endomyocardial biopsy [25, 26]. The immunofluorescence method
is performed on the fresh frozen tissue sections; therefore, the obvious
disadvantage is the inability to use this method in incidentally detected
amyloid in biopsy specimens, which are formalin-fixed paraffin embedded. A
panel of antibodies marked with fluorochrome applied on fresh frozen tissue
sections differs according to established practice of individual institutions.
Polarization and fluorescence results should be correlated with the bright-
field appearance in order to avoid pitfalls (for example red blood cells,
collagen, etc.).
ELECTRON MICROSCOPY, X-RAY FIBRE DIFFRACTION
Examination of ultrathin sections of tissues containing amyloid using
electron microscopy shows the unique architecture – the structure of rigid,
non-branching, randomly oriented fibrils with an average diameter of 7 – 12
nm. Each fibril is composed of four to six twisted protofibrils [3, 18].
Pavla Flodrova, Tomas Pika and Patrik Flodr
84
In addition to amyloid, there are several more fibrillary-organized deposits
with their own characteristics, for example in immunotactoid
glomerulonephritis, light chain deposition disease, diabetic nephropathy, lupus
nephritis, and others [18, 27].
Every type of tissue is acceptable for ultrastructural evaluation including
abdominal fat tissue; however, it is not recommended to use the electron
microscopy in a routine clinical practice.
Small biopsy tissue samples for the electron microscopy are mostly
processed fresh without fixation and embedding into paraffin, however
occasionally it is possible to use stored archival material of FFPE tissues.
A tissue specimen is usually fixed in modified Karnovsky’s solution and
embedded in epoxy resin. Ultrathin sections are subsequently stained using
Reynold’s solution.
Modification of the electron microscopy in order to shift from diagnostic
to typisation of amyloid is immuno-electron microscopy which combines
immunohistochemistry and the electron microscopy. High resolution electron
microscopic demonstration of antigens is visible within the cells, on the
surface of the cell or in the interstitium. The basic building protein of amyloid
is characterised by specific gold-labeled secondary antibodies bound to
monoclonal and polyclonal primary antibodies against κ and λ
immunoglobulin light chains and other amyloidogenic proteins. Using this
approach the antibody is colocalised within the amyloid fibrils and this fact
reduces the background staining which is connected with nonspecific staining
in standard immunohistochemistry [18, 28]. Preembedding or postembedding
immunogold labelling are two distinct techniques that are available depending
on whether the immunolabeling comes before or after embedding the sample
in resin. Like other antigen-antibody based reactions, imunnoelectrone
microscopy has also some limitations arising from changes of antigen during
tissue processing.
This time-consuming method is only available in highly specialised
centres with adequate technical equipment; however it can contribute to solve
difficult cases. When applied on abdominal fat samples, these techniques are
effective for determination of amyloid deposits in patients with clinical and
laboratory mediated suggestion of amyloidosis [29].
Structure-oriented knowledge of amyloid characterized by high-resolution
structural study using X-ray fibre diffraction shows characteristic pattern with
a meridional reflection at ~4.7 Å corresponding to the inter-β strand spacing
and an equatorial reflection at ~6-11 Å corresponding to the distance between
stacked β sheets [30].
Tissue Examination in a Diagnostic Procedure
85
MASS SPECTROMETRY-BASED METHODS
To avoid indirect evidence of the basic building protein, which is
sometimes problematic in immune-based techniques, a new methodology in
the proteomic analysis has been established. Mass spectrometry-based (MS)
methods are promising diagnostic trend with direct protein recognition from a
minimal amount of tissue sample, moreover it almost excludes subjectivity of
the human factor. For these reasons, this type of the proteomic analysis ceases
to be a research method and becomes an important tool for amyloid
determination in diagnostic centres around the world. MS-based methods are
developed for FFPE tissues, tissue sections from frozen tissue and fresh
adipose tissue. Acouple of relevant high-throughput methods are laser
microdissection-liquid chromatography/mass spectrometry (LMD-LC/MS)
and multidimensional protein identification technology (MudPIT) based on
2D-MS/MS, both of them bring results in identification of basic
amyloidogenic protein [20, 31], however they require a different interpretation
and also a preanalytical process in sample preparation and protein separation
[20]. The great advantage of majority of MS-based methods is the possibility
to use archival FFPE tissue samples, which can be forwarded to consulting
institution, especially in case of incidental finding of amyloid in a biopsy
specimen.
Proteins which can generate amyloid deposits are also generally present in
tissues and in blood; therefore the examination of whole tissues/tissue sections
is not appropriate. Specific sampling technique – laser microdissection (LMD)
– significantly increases the specificity by removal of overwhelming majority
of the other non-amyloid components of the tissue. FFPE sample sections are
in most cases placed on special slides or membranes and stained to visualise
amyloid (usually using Congo red). Amyloid is then cut by laser, moved from
the slide or membrane and collected into adequate test-tubes.
A common part of MS-based procedure is that the amyloid/tissue sample
undergoes protein extraction and digestion of proteins into individual peptides
by trypsin, followed by liquid chromatography separation and detection using
mass spectrometry. A high-tech software processing is required. As a result, a
list of proteins based on peptides identified by MS is generated; each list
contains a complete spectrum of detected proteins in the sample. Sorting
according to their abundance or according to the association with amyloid is
commonly used. During software processing it is possible to analyse the
results with regard to the mutant forms of inherited variants of amyloid,
Pavla Flodrova, Tomas Pika and Patrik Flodr
86
allowing a prediction of the specific amyloidogenic mutation. Determination
of the mutation on the DNA level is a mandatory consecutive step.
Using fat tissue samples obtained by fine-needle aspiration from umbilical
region carries a slightly different practice within the methodology, which does
not require laser microdissection in sample proceeding of the adipose tissue. In
comparison with examination of FFPE tissues the specificity is similar but the
sensitivity is slightly reduced (88%); this is explained by uneven distribution
of amyloid, which complicates tissue processing [11]. One sample is divided
into two parts of which one is used for MS-based analysis and the second part
is Congo red-stained.
There are several questionable points known in the proteomic analysis, for
example some proteins lack cleaving sites for trypsin, therefore no peptides for
LC/MS detection originate. Other important part is software processing with a
source of publicly available peptide databases which may omit peptides
generated by for example germline polymorphisms.
As a consequence of the above-mentioned facts, single proteomic mass
spectrometry-based test can replace many immunohistochemical tests on many
tissue sections in searching for all currently known amyloid subtypes [10]. The
size of biopsy samples is often very limited for cutting histological sections,
especially in cases of fine-needle biopsies. If an invasive procedure is
indicated such as a kidney biopsy, we need a reliable diagnostic tool.
Sensitivity and specificity of LMD-LC/MS method is very high even in the
cases of rare hereditary variants and reaches almost 100% [10, 11].
New MS-based approach called matrix-assisted laser desorption/ionization
(MALDI) imaging mass spectrometry (IMS) has been established recently
[32] and it is suggested for investigation of amyloid deposits in-situ without
the need of time-consuming tissue microdissection and protein extraction [33].
Besides the purpose of the diagnostic test, detailed knowledge of the
proteome of amyloid deposits helps elucidate the pathogenesis of the disease
and describe the new variant of amyloid.
FAT TISSUE ASPIRATES – CYTOLOGICAL
EXAMINATIONS OF THE SMEARS
Fat tissue aspiration brings several benefits in comparison with a standard
biopsy. The location of the tissue collection – anterior abdominal wall in the
umbilical region – is easily accessible. The process is low cost and minimally