Unreliability of immunohistochemistry for typing amyloid deposits.
Article Type: Letter to the editor
Authors: Solomon, Alan
Murphy, Charles L.
Westermark, Per
Pub Date: 01/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: Jan, 2008 Source Volume: 132 Source Issue: 1
Accession Number: 230246964
Full Text: To the Editor.--The article by Satoskar et al (1) addresses a seminal issue; namely, the therapeutic necessity of identifying the protein that forms renal amyloid deposits. These authors state that "immunofluorescence staining for immunoglobulin light chains on renal biopsy, as the first step to differentiate between AL [primary or light chain-associated] and AA [secondary or serum amyloid A protein-associated] amyloidosis, may sometimes be inconclusive or even misleading." Notably, their commercially obtained anti-light chain antibodies lacked specificity, that is, they cross-reacted with AA fibrils and did not necessarily reveal a predominant immunostaining of one light chain type ([kappa] vs [lambda]). In an accompanying editorial, Picken and Herrera (2) discuss the numerous limitations and pitfalls that can occur when using immunohistochemistry to establish the type of amyloid present in biopsy specimens and emphasize that this method requires special expertise and reagents; furthermore, "misinterpretation may have profound consequences."

These reports indicate the danger of relying on immunohistochemical methods, as well as ancillary laboratory or clinical data, to ascertain if an individual has AL, AA, or some other kind of amyloid disease. To obtain an accurate diagnosis, the amyloid contained in biopsy-derived specimens are best extracted and analyzed chemically. (3) In this regard, we have used tandem mass spectrometry to gain this information from formalin-fixed, paraffin-embedded tissue biopsies, as well as from subcutaneous fat aspirates, (4) and perform these studies on request. Depending on the size of the biopsy specimen and amount of amyloid present, as few as four to six 4-[micro]m-thick sections may suffice (even if there is no tissue remaining in the paraffin blocks, it is possible to use sections that have been subjected to Congo red, hematoxylin-eosin, or other histochemical stains). Further, material scraped from 1 or 2 amyloid-containing fat aspirate smears have also yielded sufficient fibrils for tandem mass spectrometry analysis.

Because the treatment as well as prognoses of patients with amyloidosis is dependent on the amyloid type, it is crucial that the nature of the fibrillar protein be established unequivocally to avoid inappropriate and costly therapy that can have dire and possible legal consequences.

Alan Solomon, MD

Charles L. Murphy, MS

Human Immunology and Cancer Program

Department of Medicine

university of Tennessee

Graduate School of Medicine

Knoxville, TN 37920

Per Westermark, MD, PhD

Genetics and Pathology

Uppsala University

Uppsala, Sweden

(1.) Satoskar AA, Burdge K, Cowden DJ, Nadasdy GM, Herbert LA, NadasdyT. Typing of amyloidosis in renal biopsies: diagnostic pitfalls. Arch Pathol Lab Med. 2007;131:917-922.

(2.) Picken MM, Herrera GA. The burden of "sticky" amyloid: typing challenges. Arch Pathol Lab Med. 2007;131:850-851.

(3.) Murphy CL, Eulitz M, Hrncic R, et al. Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens. Am J Clin Pathol. 2001;1 16:135-142.

(4.) Murphy CL, Wang S, Williams T, Weiss DT, Solomon A. Characterization of systemic amyloid deposits by mass spectrometry. Methods Enzymol. 2006;412:48-62.

The authors have no relevant financial interest in the products or companies described in this article.

In Reply.--We completely agree with the letter of Solomon et al. Typing of amyloidosis based on immunofluorescence and immunohistochemistry can sometimes be equivocal, and in such instances, more precise chemical or molecular methodologies should be performed, if possible. unfortunately, the diagnostic renal or other organ biopsy for amyloidosis is usually small and by the time all the appropriate diagnostic sections have been cut, the tissue remaining in the paraffin block is frequently not sufficient to perform the tests that Dr Solomon's laboratory is offering. Fortunately, in most instances, the immunohistochemical diagnosis is straightforward; however, in the cases in which the diagnosis is equivocal, a repeat biopsy specifically for chemical or molecular testing could be considered. Such diagnostic tests have to be appropriately validated, highly sensitive, and specific and have a quick turnaround time.

Tibor Nadasdy, MD

Anjali A. Satoskar, MD

Daniel J. Cowden, MD

Gyongyi M. Nadasdy, MD

Department of Pathology

Kelly Burdge, MD

Lee A. Hebert, MD

Department of Internal Medicine

Division of Nephrology

The Ohio State university

Columbus, OH 43210

The authors have no relevant financial interest in the products or companies described in this article.

In Reply.--The letter by Solomon et al underscores the importance of correct diagnosis of amyloid type in patient management. The authors also state that, given its limitations, immunohistochemistry is unreliable for the typing of amyloid deposits. The authors conclude that the only reliable method is one that is based on direct chemical identification of the amyloid protein.

Chemical methods allowing direct molecular identification of the amyloid protein have recently been developed in highly specialized research laboratories (recently cited in Picken et al (1)). By using proteomics technologies, the amount of tissue needed is much reduced, thus making it possible to apply these methods to clinical biopsies. Although these are welcome and much-needed major advances in the field of amyloidosis diagnosis, there are issues that are relevant to their clinical application that need to be considered. (1) These new technologies have not yet been validated in large numbers of samples at multiple centers. (2) Moreover, the standards for interpretation of mass spectrometry results need to be evaluated and published by the Association of Biomolecular Resource Facilities. There is an urgent need to address regulatory issues such as accreditation, licensing, validation, and the analyte-specific reagent status of these emerging technologies. To guarantee accurate results, other crucial issues need to be considered and addressed, including specifics regarding the sample, the cost of providing the service, and turnaround time.

The question arises whether amyloid proteomics should replace or complement current immunohistochemistry methods. Well-performed immunohistochemistry can be reliable in a significant percentage of cases. (3) In this regard, we agree with Nadasdy et al in the accompanying reply that, at least for now, in cases in which the immunohistochemistry diagnosis is straightforward, it continues to be a clinically useful and very practical technique. It is, however, important that the study and the interpretation of results be performed carefully and with an awareness of its limitations. In particular, the use of adequate sample (preferably fresh tissue) and observer experience are important. Nonetheless, as we stated previously, in cases that are inconclusive or negative, evaluation by a reference laboratory with the capability of applying more sophisticated methods should be pursued. (4) Although the situation may change in the future, in current pathology practice the molecular characterization of amyloid proteins represents a valuable complement rather than a direct replacement of immunohistochemistry.

In view of the dramatic changes taking place in the management of patients with amyloidosis, pathologists need to be an integral part of collaborative efforts, led by the International Society of Amyloidosis, to develop recommendations and standards for the clinical diagnosis of amyloidosis. This work is in progress and it is hoped that it will result in definitive recommendations as to how to handle amyloidosis cases from a pathology perspective. However, we have to be prepared to adjust these recommendations as the new technologies emerge and become validated.

Maria M. Picken, MD, PhD

Department of Pathology

Loyola University Medical Center

Maywood, IL 60153

Guillermo A. Herrera, MD

Department of Pathology

St Louis University

St Louis, MO 63104

(1.) Picken MM, Hazenberg BPC, Obici L. Report from the diagnostic interactive session. In: Skinner M, Berk JL, Connors LH, Seldin DC, eds. XIth International Symposium on Amyloidosis. Boca Raton, Fla: CRC Press; 2007:377-382.

(2.) Comenzo RL. Constraints on the pursuit of diagnostic confidence. Blood. 2006;108:776 777.

(3.) Picken MM. Immunoglobulin light and heavy chain amyloid: renal pathology and differential diagnosis. Contrib Nephrol. 2007;153: 135-155.

(4.) Picken MM, Herrera GA. The burden of "sticky" amyloid: typing challenges. Arch Pathol Lab Med. 2007;131:850-851.

The authors have no relevant financial interest in the products or companies described in this article.
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