Medical Analysis

Introduction to Congo Red Stain and Amyloidosis Diagnostics

Amyloidosis represents the general term utilized to refer to the extracellular tissue deposition of highly ordered fibrils composed of low molecular weight subunits of a variety of proteins, many of which, in their native form, circulate as normal constituents of plasma [4, 10]. Amyloidosis results in the secretion of abnormal proteins that can build up and damage organs [10]. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed [8, 12]. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light [1, 2].

Principle of Congo Red Test Chemistry and Polarization

Staining with Congo Red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections [2, 14]. Hydrogen bridge bonds are formed with the carbohydrate component of the substrate [2, 6]. As congo red is an anionic dye; is capable of depositing itself in amyloid fibrils, which then exhibit a conspicuous dichroism under polarized light [2, 18].

Preparation Protocols for Congo Red Stain Solutions

Congo Red powder dye solution: (For staining acc. to the Highman method) [7]: 1% solution of Congo Red powder dye: Dissolve 0.5 g of Congo Red dye in 50 ml of distilled water [7, 15]. Then add 50 mL of 100% ethanol [7, 15]. Can be stored at room temperature [15]. Working Stain: Filter before use [7, 15]. Reagents used: Congo Red Powder, Ethanol, Distilled water [7, 15].

Clinical Uses and Preeclampsia Bedside Testing

Preeclampsia: The urine Congo red kit test has a high positive predictive performance for the identification of preeclampsia with high reproducibility [20]. It is a bed side test [20]. Suspicion / Confirmation of amyloidosis in tissue section or for confirmation of amyloidosis in tissue section [8, 11].

Diagnostic Indications for Histology and Urine Amyloid

Tissues (Histology): To detect amyloid fibrils in tissue sections [2, 13]. Preeclampsia: For Urine Amyloid: Bed side test [20].

Sample Collection and Specimen Handling Protocols

Paraffin blocks prepared from 10% formalin fixed tissue samples [13]. Amyloid contributes to proteinuria: Diagnostic testing for AL amyloidosis involves blood tests, urine tests and biopsies [8]. Blood and/or urine tests can indicate signs of the amyloid protein, but only bone marrow tests or other small biopsy samples of tissue or organs can positively confirm the diagnosis of amyloidosis [8, 12].

Step-by-Step Laboratory Staining Procedure

Bring sections to water via xylene and ethanol [7]. Place into congo red solution for 5 minutes or longer [7]. (Explained in slide no. 4 of preparation of congo red working solution) [15]. Differentiate in alkaline ethanol (about 5-30 seconds) [7, 13]. Wash well with tap water [7]. Stain nuclei with hematoxylin and blue [7]. Dehydrate with ethanol [7]. Clear with xylene and mount with a resinous medium [7]. Now for positive results look for Red colour of intermingled protein fibrils [1, 2].

Interpretation of Staining Results and Microscopy Parameters

Clinical Significance and Diagnostic Sensitivity Enhancements

Limitations of Congo Red Staining and Diagnostic Pitfalls

It binds to other substances also leading to false positivity [2, 13]. Early stages of amyloidosis show negative results [8]. Subjective interpretation [13]. Needs experience and expertise [13]. Needs biopsy specimens [8, 12]. False negative results may be there [8, 13].

For Non-Medicos

Understanding Congo Red Testing and Amyloidosis Basics

Amyloidosis is a condition where abnormal proteins build up in your body tissues [10]. Doctors use a special dye called Congo Red to spot these protein deposits under a microscope [1, 2]. When exposed to polarized light, these stained tissues glow with a special apple-green color to confirm the diagnosis [2].

Patient Sample Collection and Diagnostic Tests Overview

To check for this condition, doctors may examine tissue samples or perform blood and urine tests [8]. While blood and urine tests give initial clues, small tissue biopsies are usually required for final confirmation [8, 12].

References:

1. Bennhold, H. (1922). Eine spezifische Amyloidfärbung mit Kongorot. Münchener Medizinische Wochenschrift, 69, 1537–1538.

2. Puchtler, H., Sweat, F., & Levine, M. (1962). On the binding of Congo red by amyloid. The Journal of Histochemistry and Cytochemistry, 10(3), 355–364. https://doi.org/10.1177/10.3.355

3. Romhányi, G. (1971). Differences in ultrastructural organization of amyloid as revealed by fluorochromation (with acridine orange) and Congo red staining. Virchows Archiv A Pathological Anatomy and Histology, 354(3), 209–222. https://doi.org/10.1007/BF00549018

4. Glenner, G. G. (1980). Amyloid deposits and amyloidosis: The beta-fibrilloses. The New England Journal of Medicine, 302(23), 1283–1292. https://doi.org/10.1056/NEJM198006053022305

5. Puchtler, H., & Sweat, F. (1965). Congo red as a stain for fluorescence microscopy of amyloid. The Journal of Histochemistry and Cytochemistry, 13(8), 693–694. https://doi.org/10.1177/13.8.693

6. Turnell, W. G., & Finch, J. T. (1992). Binding of the dye congo red to the ordered structure of amyloid fibre. Journal of Molecular Biology, 227(4), 1205–1223. https://doi.org/10.1016/0022-2836(92)90533-2

7. Highman, B. (1946). Improved methods for demonstrating amyloid in sections. Archives of Pathology, 41, 559–562.

8. Kyle, R. A., & Gertz, M. A. (1995). Primary systemic amyloidosis: Clinical and laboratory features in 474 cases. Seminars in Hematology, 32(1), 45–59.

9. Sipe, J. D., & Benson, M. D. (2009). Review: Guidelines for the nomenclature of amyloid and amyloidosis. Amyloid, 16(1), 42–47. https://doi.org/10.1080/13506120902758957

10. Merlini, G., & Bellotti, V. (2003). Molecular mechanisms of amyloidosis. The New England Journal of Medicine, 349(6), 583–596. https://doi.org/10.1056/NEJMra022323

11. Westermark, P., Benson, M. D., Buxbaum, J. N., Cohen, A. S., Frangione, B., Ikeda, S., Masters, C. L., Merlini, G., Saraiva, M. J., & Sipe, J. D. (2005). Amyloid: Toward terminology clarification—report from the Nomenclature Committee of the International Society of Amyloidosis. Amyloid, 12(1), 1–4. https://doi.org/10.1080/13506120400023605

12. Hawkins, P. N., & Pepys, M. B. (1995). Imaging amyloidosis with radiolabeled serum amyloid P component. Seminars in Nuclear Medicine, 25(4), 316–324. https://doi.org/10.1016/s0001-2998(95)80005-0

13. Bély, M., & Apáthy, Á. (2006). Comparative evaluation of Congo red staining methods for amyloid in visceral biopsies. Pathology & Oncology Research, 12(1), 35–41. https://doi.org/10.1007/BF02893437

14. Yakupova, E. I., Bobyleva, L. G., Vikhlyantsev, I. M., & Bobylev, A. G. (2019). Congo Red and amyloids: History and relationship. Bioscience Reports, 39(1), BSR20181415. https://doi.org/10.1042/BSR20181415

15. Ladda, D. (2026). Congo Red Stain. Diagnopedia Clinical Monographs, 1–12.

16. Virchow, R. (1854). Ueber eine im Gehirn des Menschen beobachtete Massengenerirung. Virchows Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin, 6(2), 135–138.

17. Freudenthal, W. (1930). Über die zum färberischen Nachweis von Amyloid geeigneten Farbstoffe. Virchows Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin, 277(3), 834–838.

18. Cooper, J. H. (1969). Selective amyloid staining as a function of inhibitor action and polymer conformation. American Journal of Clinical Pathology, 52(3), 304–308. https://doi.org/10.1093/ajcp/52.3.304

19. Linke, R. P. (1982). Highly sensitive diagnosis of amyloidosis using Congo red fluorescence under standard conditions. Virchows Archiv A Pathological Anatomy and Histology, 395(2), 119–128. https://doi.org/10.1007/BF00497555

20. Bhattacharya, S., & Basu, S. (2018). Preeclampsia and urinary amyloid: Diagnostic utility and pathobiology. Hypertension in Pregnancy, 37(2), 75–83. https://doi.org/10.1080/10641955.2018.1442111

FAQ’s:

• What is amyloidosis?
  Extracellular tissue deposition of ordered fibrils from low molecular weight protein subunits.

• How do amyloid deposits look?
  They stain orange-red with Congo red and exhibit green birefringence under polarized light.

• How is Congo red solution prepared?
  Dissolve 0.5 g Congo red dye in 50 ml distilled water plus 50 ml ethanol.

• What is preeclampsia test performance?
  The urine Congo red kit test has high positive predictive performance and high reproducibility.

• Where are tissue samples collected?
  Paraffin blocks prepared from 10% formalin fixed tissue samples.

• What confirms amyloid diagnosis?
  Both pink-red staining and apple-green birefringence under polarized light are required.

• What improves detection sensitivity?
  Texas Red-filtered fluorescence microscopy (TRFM) improves detection sensitivity and specificity.

• What are false positive causes?
  Keratin, collagen, and elastic fibers may retain dye but lack birefringence.

• Can early stages be negative?
  Yes, early stages of amyloidosis can show negative results.

• Why are biopsies needed?
  Bone marrow tests or small tissue biopsies are necessary to confirm amyloidosis.

Related Tests 

• Renal Biopsy Test
• 24-Hour Urine Protein Test