Why congo red stain

A common clinical analyzer has a polarizing filter with a built-in compensator which usually adds color and is often used for crystal examination. The use of such an analyzer in examining Congo red stained specimens, frequently leads to a field that is excessively bright and sometimes forces the observer to partially uncross the analyzer in order to accentuate the apple green birefringence.

However, this results in appearance or enhancement of a bluish-green hue Fig. 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. In this report, we show that suitable microscopy equipment can increase the sensitivity of identifying the amyloid-specific birefringence in Congo red-stained tissue sections. Early diagnosis of systemic amyloidosis is essential to reducing morbidity and mortality of the disease. Despite the seriousness of the disease and the benefit of early detection, an accurate pathologic diagnosis is still challenging.

Spotty nature of disease, variation in organ-to-organ of the density of amyloid deposits, and the difficult of reproducible tissue staining, all increase the odds of false negative and false positive results.

A negative tissue pathology report can effectively exclude the diagnosis of amyloidosis, which is then frequently never reconsidered among differential. The standard of care for multiple myeloma, mono, and polyclonal gammopathy is conservative follow up, unless there is identifiable end-organ damage or amyloidosis [ 18 ].

Accordingly, missing an early diagnosis of amyloidosis can deprive a patient from receiving lifesaving treatment and can lead to costly and sometimes invasive investigations to pursue alternative diagnoses.

In the case of transthyretin or fibrinogen amyloidosis, early liver transplantation usually arrests disease progression and can even be curative [ 19 , 20 , 21 ]. Therefore, even a marginal improvement in sensitivity of detection of amyloid in tissue specimens will help in assuring that patients with this serious and frequently fatal disease can be treated promptly and receive accurate prognostic information.

The real prevalence of amyloidosis is not known. The prevalence of multiple myeloma is dwarfed by the prevalence of monoclonal gammopathy, which can be as high as 8. When the diagnosis is missed discipline-specific bias leads to ascribing to organ dysfunction to diabetes in case of renal disease and neuropathy and cardiac symptoms on hypertension or ischemia.

Yet, there is no systematic data that examine the accuracy of these presumptive etiologies, and it is not inconceivable that some fraction of these patients may be incorrectly classified. Owing to the patchy nature of amyloidosis, especially during its early stages, amyloid deposition could be restricted to just a small area of the tissue biopsy only visible at a limited angle of slide viewing. Thorough examination of each section using a mechanical rotating stage to view slides at variable angles is essential to avoid missing such deposits.

We also recommend that plastic cover slips be avoided as they can interfere with the ability to perform crossed polarized light examination and reduce ability to identify subtle or low density amyloid deposits. Low density deposits are enough to make the diagnosis due to the patchy nature of the disease. When a sample is deemed negative or equivocal, there is a need to follow previous published modifications like the use of polar mounting media or omitting the alcohol differentiation step when examining collagen rich tissue to avoid interference [ 26 , 27 , 28 , 29 ].

Finally, the use of proper optics like those of a metallurgical microscope is essential to avoid missing the presence of small deposits of amyloid in Congo red-stained tissue.

There is variability on the reporting of Congo red stained slides between different labs and pathologists. We identified important pearls that can improve the ability to identify amyloid material in Congo red stained tissues. We found that it is critical to use microscope with proper strain free optics and avoid the use of polarizer with built-in compensator.

The use of mechanical rotating stage will reduce the chance of missing subtle or low-level amyloid deposits which can only produce birefringence at specific angles. Last, plastic cover slips can lead to inability to examine the slides under crossed polarized light.

Improving sensitivity of the Congo red evaluation can aid in early diagnosis of amyloid and will have tremendous impact on clinical outcome of some patients. Kyle RA. Amyloidosis: a convoluted story. Br J Haematol. Amyloid fibril proteins and amyloidosis: chemical identification and clinical classification International Society of Amyloidosis nomenclature guidelines.

Current trends in diagnosis and Management of Cardiac Amyloidosis. Curr Probl Cardiol. Article Google Scholar.

Primary systemic amyloidosis: multivariate analysis for prognostic factors in cases. Nationwide biopsy survey of renal diseases in the Czech Republic during the years J Nephrol. Amyloid neuropathies. Mt Sinai J Med. Curr Opin Neurol. Sustained improvement in cardiac function with persistent amyloid deposition in a patient with multiple myeloma-associated cardiac amyloidosis treated with bortezomib.

Int J Hematol. Clin J Am Soc Nephrol. AL-amyloidosis is underdiagnosed in renal biopsies. Nephrol Dial Transplant. Renal pathologic spectrum in an autopsy series of patients with plasma cell dyscrasia. Arch Pathol Lab Med. PubMed Google Scholar. Bely M, Makovitzky J.

More recently, Clement and Truong have shown how fluorescence microscopy using a Texas red filter may enhance the identification of amyloid deposits in Congo red-stained tissue sections, especially when deposits are small. Steensma DP. Arch Pathol Lab Med. Griesbach, H. Bennhold, H. Design: Primary sources were consulted extensively, including 19th-century corporate documents, newspapers, legal briefs, patents, memoirs, and scientific papers.

Setting: Sources were obtained from multiple university libraries and German corporate archives. Atomic force microscopy This technique can be used to investigate the size and morphology of protein aggregates in solution [ ]. No No Yes Yes When contact mode is used, high shear forces cause damage to the fibrils and may require immobilisation strategies [ ]. For the tapping-mode in liquid, current scan speeds are too slow to image rapid processes, and often, samples fibrils need to be well-adhered to a surface.

Other atomic force microscopic techniques, including the use of support surfaces e. Dynamic light scattering Dynamic light scattering is a laser scattering technique capable of unbiased analysis of size distributions for diffusing particles in the nanometre to micrometre size range [ ]. The ability to resolve multimodal size distributions and make absolute size measurements makes dynamic light scattering a powerful technique for systems with heterogeneous species.

It has been used to quantitatively study fibril formation in a range of systems from different proteins [ — ]. No No No Yes A hydrodynamic radius of the particle is measured, but this can differ from the true radius. As a result, determination of the real size and shape of macromolecules is difficult. Fluorescence correlation spectroscopy This highly sensitive analytical technique is used to measure dynamic molecular parameters, such as diffusion time from which particle size can be calculated , conformation and concentration of fluorescent molecules [ , ].

It has been particularly powerful for characterising size distributions in molecular associations e. Fluorescence correlation spectroscopy could therefore be used as a highly sensitive and specific competition assay to identify potential inhibitors of fibril formation [ ]. No No No Yes This technique requires the use of fluorescent tags to label amyloid samples.

This makes it difficult and rarely used. Analytical size-exclusion chromatography The method is used to separate a diverse range of differently sized particles by passing a solution containing the particles through a partially permeable gel medium. It is used to analyse intermediates mainly, oligomers and identify soluble aggregates in tissue [ , ].

No No No Yes It is impossible to study aggregates with high molecular weight. This method is inefficient for scale-up because size-exclusion chromatography performs poorly on large liquid volumes. Analytical ultracentrifugation This technique is based on the sedimentation velocity analysis used to determine the size, shape, and hydrodynamic behaviour of soluble macromolecules, including amyloid fibrils, as well as to study the process of amyloid aggregation [ — ].

No No No Yes This technique is used only for specific tasks. Open in a separate window. CR dye Dyes are usually aromatic, heterocyclic compounds, some of which are toxic and possibly carcinogenic [ ].

Table 2 Relation between different dyes and selective staining of amyloid by [ ] with our modifications. The use of CR in the study of amyloidosis As mentioned above, the use of CR for amyloidosis identification began with experiments performed by Bennhold [ 14 ].

The binding of CR to amyloids CR was the first direct dye for cotton, which did not require a mordant or chemical to fix the colour to the material [ ]. Figure 1. The main hypothetical models of the binding of CR to amyloids A Dye binding mediated by hydrogen bonding between primary hydroxyl groups of the peptide chain similar to the polysaccharide chain and the amino groups of CR Puchtler et al. Exceptions to the rules There are facts that indicate histochemical staining with CR is non-specific.

Figure 2. Conclusion CR, now a classic dye, has played a role in the history of amyloid research. Competing interests The authors declare that there are no competing interests associated with the manuscript. Funding This work was supported by the Russian Foundation for Basic Research [grant number ]; and the Russian Science Foundation [grant number ]. References 1. Chiti F. Fowler D. PLoS Biol. Romling U. Claessen D. Genes Dev. Otzen D. Life Sci. L Hydrophobins, the fungal coat unraveled.

Acta , 79—86 Iconomidou V. Slotta U. Kyle R. Scleiden M. Klassische Schriften zur Zellenlehre. Schmidt C. Friedreich N. Fonteyn N. Bennhold H. Elghetany M. Stain Technol. Astbury W. Eanes E. Bonar L. Maji S. FEBS Lett. Cohen A. Nature , — Jimenez J. EMBO J.

Petkova A. Jaroniec C. Goldsbury C. Harper J. Walsh D. Huang T. An in vitro model of preamyloid. Kumar S. Berthelot K. Yakupova E. Prusiner S. Science , — Horwich A. Cell 89 , — Cell 93 , — Kushnirov V. Prion 1 , — Cell , — Dorta-Estremera S. Goldschmidt L. Galvin J. Methods Mol. Flodrova P. Pathology 50 , — Strege R. Report of 43 cases in an unselected autopsy series.

De Genst E. Acta , — [ PubMed ] [ Google Scholar ]. Higaki J. Amyloid 23 , 86—97 Kayed R. Methods Enzymol. Murakami K. Sarsoza F. Acta Neuropathol. Dumoulin M. Biochimie 86 , — Fang X. Neuroimage , — Rasmussen J. Aslund A. ACS Chem. Psonka-Antonczyk K. Westermark G. Frid P. Brain Res. Meehan S.

Linke R. Protein Reviews Uversky V. Howie A. Amyloid 17 , — Puchtler H. Persichilli C. Lindberg D. Biochemistry 56 , — Naiki H. Cohen S. Groenning M. Vassar P. LeVine H. Lindgren M. Maezawa I. Grudzielanek S.

McCubbin W. Abe H. Scarlett G. Calero M. In Amyloid Proteins. Smith B. Stuard B. Bouchard M. Protein Sci. Zou Y. B , — Karamanos T. Tycko R. Scheidt H. Aimoto S. Lansbury P. Jr, Costa P. Burkoth T. Balbach J. Antzutkin O.