Histochemical Differential Diagnosis and Polarization Optical Analysis of Amyloid and Amyloidosis

Amyloidosis is characterized by extracellular deposition of protein fibrils of chemically heterogeneous composition. Early recognition and identification of amyloid deposits allows an early start of therapy, which may entail a better prognosis. Congo red staining according to Romhányi (1971) is a highly specific and sensitive method for early microscopic recognition of amyloidosis. The main and most important types of amyloidosis may be distinguished by classic histochemical methods of performate pretreatment according to Romhányi (1979), or by KMnO4 oxidation according to Wright (1977) followed by Congo red staining and viewed under polarized light. Differences in the speed of breakdown (disintegration) of amyloid deposits according to Bély and Apáthy allow a more precise distinction of various types of amyloid.


INTRODUCTION
Amyloidosis is characterized by extracellular deposition of protein fibrils of chemically heterogeneous composition. The classification of amyloid is based on the types of amyloid fibril proteins and their precursors [1,2]. Several diseases or disorders may be complicated by systemic or localized deposition of amyloid proteins (Table 1.).
These amyloid deposits are different, depending on (a) the type and (b) the stage of amyloidosis [3,4].
The aim of the present study was to demonstrate, by the classical histochemical methods of Romhányi [5] or Wright [6], the differentiation of systemic and localized amyloidosis using Congo red staining [7] after degradation of deposited amyloid according to Bély and Apáthy [8,9].

MATERIAL AND METHODS
Fifty-seven systemic cases of systemic secondary AA amyloidosis were studied in a randomized autopsy population of 308 in-patients with various autoimmune diseases. Amyloid A protein deposits were present in 52 of 234 patients with rheumatoid arthritis (RA), in 2 of 50 with systemic lupus erythematosus, in 2 of 12 with psoriatic arthritis, and in 1 of 12 with progressive systemic sclerosis. The patients died at the National Institute of Rheumatology between 1970 and 1999. Systemic immunoglobulin light-chain (AL-λ, or κ) amyloidosis was studied in a selected autopsy population of 5 in-patients with B-cell lymphoma. Systemic senile amyloidosis with amyloid transthyretin deposits (ATTR) was found and analyzed in an 85-year-old autopsy patient, and systemic β2-microglobulinrelated amyloidosis (Aβ2M), associated with hemodialysis was in a further one [10]. Isolated cerebral amyloid β protein deposits (Aβ) wer studied in a selected autopsy population of 12 in-patients with Alzheimer's disease, who died at the National Institute of Psychiatry and Neurology [11].
Regional, exclusively mediastinal amyloid A protein (AA) deposits were detected with bronchioloalveolar carcinoma in one of 234 RA patients. We found renal retention cysts containing concentrated β2microglobulin glomerular filtrate (localized-Aβ2M) in 2 of 52 RA patients in combination with coexistent AA amyloidosis [4]. Islet amyloid polypeptide (AIAPP) localized to the islets of Langerhans were present in 16 of 101 pancreas, corpus amylaceum of the lung in 8 of 169, and corpus amylaceum of the prostate in 7 of 13 RA autopsy patients. Other forms of tissue (organ)-limited, localized amyloids were sporadically found in routine biopsy material.
The tissue specimens were fixed in 8% formaldehyde and embedded in paraffin. Serial sections were cut and stained with Hematoxylin and Eosin or Congo red according to , without alcoholic differentiation, and sealed with gum arabic.

RESULTS
The histochemical characteristics of systemic and localized amyloidosis are summarized in Table 2.

• The oriented array (arrangement, settlement) of amyloid filaments and fibrils in amyloid deposits induces a birefringence of typical apple green polarized color.
There is no pathognostic difference in color of polarized light produced by biochemically heterogeneous amyloid deposits.
• Different oxidative agents (performate, KMnO 4 , etc.) may disintegrate the oriented arrangement of amyloid filaments and fibrils; consequently, the birefringence with typical apple green polarized color decreases, or disappears, depending on the applied oxidative agents, and depending on the time of destruction.
For example, systemic or localized amyloid A deposits are very sensitive to performate pretreatment, birefringence disappears within 1 sec; on the other hand, these deposits are relatively resistant to KMnO 4 oxidation, destruction takes a longer time, birefringence disappears after 30 sec -1 min only.
• Performate is a more aggressive destructive agent than KMnO 4 oxidation. Systemic amyloid A deposits will disintegrate within 1 sec after performate pretreatment, whereas AL-λ, or κ amyloid takes more than 5 sec, Aβ2M more than 15 sec, ATTR more than 25 sec to disintegrate.
Disintegration of systemic amyloid A deposits takes at least 1 min, that of systemic AL-λ, or κ amyloid deposits at least 3 min, that of Aβ2M at least 30 sec, and disintegration of ATTR amyloid deposits takes at least 10 min of KMnO 4 oxidation.
After performate pretreatment localized amyloid A deposits will disintegrate within 1 sec, but localized AL-λ, or κ amyloid deposits are resistant to performate pretreatment for 20 sec (at least), localized Aβ2M 20 sec (at least), dystrophic articular cartilage amyloid for 3 sec.
Disintegration of localized amyloid A deposits takes at least 3 min, localized AL-λ, or κ amyloid deposits at least 5 min, and dystrophic articular cartilage amyloid deposits at least 15 min of KMnO 4 oxidation.
• Amyloid deposits are more sensitive to performate pretreatment than to KMnO 4 oxidation. The systemic AL-λ, or κ amyloid amyloid deposits are resistant to performate pretreatment for 1-5 sec, and the localized AL-λ, or κ amyloid deposits for 1-20 sec.
The systemic Aβ2M deposits are resistant to performate pretreatment for 1-15 sec, and the localized Aβ2M deposits for 1-20 sec.
Any form of Aβ2M deposits (systemic or localized) are very sensitive to KMnO 4 oxidation: systemic, or localized Aβ2M deposits disintegrate within 30 sec.
• Deposits in systemic amyloidosis are more sensitive to performate pretreatment or to KMnO 4 oxidation than localized forms of deposits.
Systemic AL-λ, or κ amyloid deposits are resistant to performate pretreatment for 10 sec (or more), while the localized AL-λ, or κ amyloid deposits are resistant to performate pretreatment for 20 sec (at least).
Systemic Aβ2M deposits are resistant to performate pretreatment for 15 sec (or more), while the localized Aβ2M deposits are resistant to performate pretreatment for 20 sec (at least).
Systemic AL-λ, or κ amyloid deposits are resistant to KMnO 4 oxidation for 3 min, while the localized AL-λ, or κ amyloid deposits are resistant to KMnO 4 oxidation for 5 min (at least).
• The minimal (early stage), "fresh" amyloid deposits are more sensitive to performate pretreatment or to KMnO 4 oxidation than massive (late stage), "old" amyloid deposits.
Applying the same destructive agent, minimal amounts of amyloid (in case of any types of amyloid) disappear earlier than massive deposits  *** AA amyloid deposits may be R/S to KMnO 4 oxidation (for 1 min) in early stage of amyloidosis.

Remarks to Table 2, and Suggested Guideline for Differential Diagnosis of Amyloidosis
• Five sections, serially cut, may be necessary for the histochemical differential diagnosis of the main types of amyloid. • The histochemical diagnosis may be confirmed by immunohistochemical reaction on additional slides. Antihuman amyloid A reaction is specific, but minimal deposits of amyloid A may be missed with it. The minimal deposits of amyloid, viewed under polarized light, by their intensive green color in a dark field are also specific, but easier to see. The immunohistochemical analysis of AL, or β2-microglobulin reaction may be difficult especially in case of minimal deposits, because of the extensive background staining. • Different types of amyloid may exist in the same patient, or side by side in the same organ, or tissue.

Suggested Schedule for Histochemical Analysis of Amyloid Deposits
1.
Step (Second slide). , in order to confirm the suspected amyloidosis.

Performate pretreatment for 1 sec
Performate pretreatment for 1 sec is a very sensitive and specific method to recognize the AA amyloidosis. Any form of amyloid A protein deposit (systemic, or regional) is very sensitive to performate pretreatment. The birefringence of amyloid A deposits immediately disappears in slides stained with Congo red and viewed under polarized light; while other forms of amyloid deposits (AL, ATTR, Aβ2M, Aβ) are resistant and retain the specific green color under polarized light.
Performate pretreatment -applying different times for degradation -would be sufficient alone to identify the various types of systemic amyloidosis. KMnO 4 oxidation presents a second independent confirmation of the diagnosis, based on performate pretreatment.
• Systemic AA amyloidosis starts in the gastrointestinal tract [12]. AA amyloidosis is a systemic, progressive, cumulative process, and is present only in the gastrointestinal tract ("regionally") in its early stage. The patient may die in this stage (because of other reasons, than amyloidosis), and the amyloidosis looks like "regional". • Regional AA amyloidosis is very rare, and the diagnosis is difficult to prove. Special local reason (in our case malignancy) is necessary and a detailed autopsy is required.

Systemic AL amyloidosis is resistant/sensitive for 5 min KMnO 4 oxidation (under polarized light
the specific green color decreases), while of β2-microglobulin deposits will be disintegrated (Aβ2M is sensitive for 5 min KMnO 4 oxidation (under polarized light the specific green color disappears).
KMnO 4 oxidation alone (without performate pretreatment) is not sufficient to differentiate between systemic AA and systemic AL amyloidosis, because the resistance (sensitivity) of AA and AL deposits may be practically the same.
• The systemic distribution of AL amyloid deposits should be confirmed by a second biopsy, for example, abdominal, subcutaneus fatty tissue biopsy (skin is often positive in systemic AL amyloidosis). • Regional AL amyloid deposits are typically localized to the respiratory tract (e.g., pharynx, trachea, bronchi) or urogenital tract (e.g., urethra), and to the skin (e.g., amyloid tumor). The localized AL deposits are more resistant to KMnO 4 oxidation (for 30 sec -10 min), than the systemic immunoglobulin light (rarely heavy) chain deposits. This is likely due to the fact that localized amyloid deposits cause of clinical symptoms only in later stages of the disease, and are diagnosed only in advanced stages of deposition. In advanced stages of amyloidosis the deposits are characterized by mature, electron microscopically closely packed, and fragmented filaments, which are more resistant to degradation [9,10].

KMnO4 oxidation for 10 min
Systemic senile amyloidosis is resistant for 10 min KMnO 4 oxidation; while systemic AL amyloid deposits are sensitive (loss of specific green color with polarized light).

CONCLUSION
Degradation of amyloid deposits by performate treatment according to  or by KMnO 4 oxidation according to , followed by Congo red staining according to , and viewed with polarized light is a practical and easy method in differentiation of most frequent types of amyloidosis. Different times of degradation according to Bély and Apáthy [8,9] allow a more precise distinction of various amyloid deposits. Amyloid deposits may be disintegrated by other oxidative agents as well, for example, by peracetate or by alkaline solutions [13].
Immunohistochemical staining of AA amyloid deposits shows a definite positive reaction for the antihuman amyloid A component. It is an excellent and reliable staining procedure.
The immunohistochemical evaluation of AL amyloid deposits may be difficult because of the intensive background staining.
Hereditary forms of amyloidosis should be excluded by medical history, or by biochemical and molecular biologic methods. (Unfortunately we have no personal experience with histochemically differentiating transthyretin amyloidosis).
The histochemical analysis (the time-dependent degradation) of amyloid deposits differentiates between early "fresh" and late stage, massive "old" amyloid deposits. Old amyloid deposits need longer time for degradation with a given oxidative agent.
None of the currently used immunohistochemical stains differentiates between early and late stage of amyloidosis.