Improving sensitivity of amyloid detection by Congo red stain by using polarizing microscope and avoiding pitfalls

Abstract

Systemic amyloidosis is a devastating group of disorders for which there is no current cure. The treatment goal is to reduce the burden of amyloidogenic protein precursors. The treatment is only effective if applied early in the disease process before significant and irreversible end organ damage has taken place. Congo red is still the standard stain used in most histopathology laboratories to identify amyloid material in tissues. The identification of Congophilic amyloid material is challenging because of multiple interfering factors. Here we describe improved sensitivity of identifying Congophilic materials in histologic sections using a metallurgical polarized microscope specifically constructed for polarized microscopy. The microscope is equipped with strain-free optics, matching polarizers, dis-integrated compensators, and a circular mobile stage. Compared to a standard clinical microscope, this setup significantly improves sensitivity of identifying amyloid material in Congo red-stained slides. We also describe the deleterious effect of plastic coverslip which can interfere with the ability to examine the slides under polarized light. We present a series of 10 different patients who had cardiac, brain, and salivary gland biopsies that were either equivocal or deemed negative using a standard clinical microscope but were positive using the equipment described above. These samples were confirmed to be positive by other methods including electron microscopy. We conclude that use of the correct equipment is needed before ruling out amyloidosis in tissue sections.

Keywords: Amyloidosis; Congo red; Metallurgical microscope; Polarized microscopy.

Fig. 1

Congo red stained salivary gland section examined under crossed polarized light from patient with AL amyloid imaged using clinical microscope (A) and same field examined using Metallurgical microscope

Fig. 2

Congo red examined with fluorescent microscope using a Texas-red filter

Fig. 3

a Negative impact of plastic coverslips in polarized microscopy, due to disorganized polarizing effect of some plastic polymers, it is difficult to obtain proper crossing of polarized light and light pass through on cover slip side (1) and proper dark field without the coverslip (2). b Negative impact of plastic cover slip in polarized microscopy showing positive control sample examined with glass cover slip (left) or plastic coverslip (right)

Fig. 4

shows a sample imaged then re-images after rotating the stage. 4A rotated 45 degrees and 4b rotated 60 degrees. The green birefringence is no longer visible as indicated by the arrows

Fig. 5

Congo red stained slide images using standard microscope with an analyzer that has a build-in compensator. Blue hue (black arrows) compared to apple green (frame arrows). Sometimes this is caused by partial uncrossing of an analyzer with built in compensator