Human Pathology (2014) 45, 2179–2180
www.elsevier.com/locate/humpath
Correspondence
Leukocyte chemotactic factor 2 amyloidosis can be reliably diagnosed by immunohistochemical staining To the editor, In contrast to the recent report by Paueksakon et al [1], we find immunoperoxidase staining for leukocyte chemotactic factor 2 (LECT2) amyloidosis to be extremely reliable. We initially validated this immunohistochemical stain using 10 cases of LECT2 amyloidosis confirmed by mass spectrometry and 25 non-LECT2 cases, including amyloid light-chain, amyloid A, and fibrinogen A alpha-chain amyloidosis. We have subsequently stained 39 additional LECT2 amyloid cases for a total of 49. In our hands, the sensitivity of the stain is 98% with a specificity of 100% for the diagnosis of LECT2 amyloidosis. The Paueksakon et al report [1] does provide an excellent opportunity to highlight that the College of American Pathology recently issued guidelines for the validation of immunohistochemical assays [2]. The consensus opinion was that each staining protocol should be validated with a minimum of 10 positive and 10 negative cases. Immunohistochemical stains for typing amyloid is a niche application of this technique, which requires a unique validation procedure. The positive and negative cases used in the validation phase should include only amyloid cases. Specifically, tissue from amyloid cases of a different type than the antibody being tested must be used as the negative control cases. In the case of LECT2 amyloidosis, the stain should be validated on cases of LECT2 amyloidosis as the positive cases and as many different non-LECT2 amyloidoses (eg, amyloid light-chain, amyloid A, and fibrinogen A alpha-chain amyloidosis) as possible for the negative cases. This validation process must be repeated in each laboratory where a stain is to be performed for diagnostic purposes. The report by Paueksakon et al [1] represents an excellent first step in validation of an antibody for amyloidosis. Moving forward by adjusting the conditions one variable at a time until a balance is achieved, such that
0046-8177/© 2014 Elsevier Inc. All rights reserved.
LECT2 amyloidosis cases are positive and the non-LECT2 are negative, will likely result in a reliable assay. The antigen retrieval step is frequently problematic for immunohistochemical assays for amyloid subtyping, as overretrieval results in a false-positive staining pattern similar to that demonstrated in the photomicrographs from Paueksakon et al [1]. Most amyloid subtypes require little-to-no antigen retrieval to elicit a diagnostic reaction [3]. In all likelihood, adjusting this step will increase the specificity of the stain without sacrificing sensitivity. Thank you. Christopher Larsen MD Nephropath Little Rock, AR 72211 E-mail address: [email protected] http://dx.doi.org/10.1016/j.humpath.2014.06.024
References [1] Paueksakon P, Fogo AB, Sethi S. Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining. HUM PATHOL 45:1445-50. [2] Fitzgibbons PL, Bradley LA, Fatheree LA, et al. Principles of analytic validation of immunohistochemical assays: guideline from the College of American Pathologists pathology and laboratory quality center. Arch Pathol Lab Med. Published online ahead of print 2014 Mar 19. [3] Kebbel A, Röcken C. Immunohistochemical classification of amyloid in surgical pathology revisited. Am J Surg Pathol 2006;30:673-83.
Leukocyte chemotactic factor 2 amyloidosis can be reliably diagnosed by immunohistochemical staining—reply The authors reply: We thank Dr Larsen for his interest in our recent publication [1]. He noted that in his experience, LECT2
2180 immunohistochemical staining is a highly sensitive and specific test for LECT2 amyloidosis, which is in contrast to our study. We agree with Dr. Larsen’s comments about the general immunohistochemical principle that the aggressive antigen retrieval step may result in false positive staining. However, the LECT2 antibody that we utilized in this study was carefully validated as optimized for our purposes. It is noteworthy that only 4 of 45 cases of amyloid light-chain and amyloid A amyloidosis were LECT2 positive, which would be very unusual in a true example of over-retrieval. While further studies are needed to confirm our initial findings (as correctly pointed out by Dr. Larsen, this will require the analysis of a larger cohort of LECT2 amyloidosis from other, more common forms of renal amyloidosis), our studies suggest that the immunohistochemical diagnosis of LECT2 amyloidosis may need to be confirmed by laser microdissection with mass spectrometry (LMD/MS) analysis of renal tissues when the staining pattern shows equivocal intensity.
Correspondence Paisit Paueksakon MD⁎ Agnes B. Fogo MD Department of Pathology, Microbiology, and Immunology Vanderbilt University Medical Center Nashville, TN 37027 ⁎Corresponding author E-mail address:[email protected] Sanjeev Sethi MD Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, MN 55905 http://dx.doi.org/10.1016/j.humpath.2014.06.026
Reference [1] Paueksakon P, Fogo AB, Sethi S. Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining. HUM PATHOL 2014;45:1445-50.
www.elsevier.com/locate/humpath
Correspondence
Leukocyte chemotactic factor 2 amyloidosis can be reliably diagnosed by immunohistochemical staining To the editor, In contrast to the recent report by Paueksakon et al [1], we find immunoperoxidase staining for leukocyte chemotactic factor 2 (LECT2) amyloidosis to be extremely reliable. We initially validated this immunohistochemical stain using 10 cases of LECT2 amyloidosis confirmed by mass spectrometry and 25 non-LECT2 cases, including amyloid light-chain, amyloid A, and fibrinogen A alpha-chain amyloidosis. We have subsequently stained 39 additional LECT2 amyloid cases for a total of 49. In our hands, the sensitivity of the stain is 98% with a specificity of 100% for the diagnosis of LECT2 amyloidosis. The Paueksakon et al report [1] does provide an excellent opportunity to highlight that the College of American Pathology recently issued guidelines for the validation of immunohistochemical assays [2]. The consensus opinion was that each staining protocol should be validated with a minimum of 10 positive and 10 negative cases. Immunohistochemical stains for typing amyloid is a niche application of this technique, which requires a unique validation procedure. The positive and negative cases used in the validation phase should include only amyloid cases. Specifically, tissue from amyloid cases of a different type than the antibody being tested must be used as the negative control cases. In the case of LECT2 amyloidosis, the stain should be validated on cases of LECT2 amyloidosis as the positive cases and as many different non-LECT2 amyloidoses (eg, amyloid light-chain, amyloid A, and fibrinogen A alpha-chain amyloidosis) as possible for the negative cases. This validation process must be repeated in each laboratory where a stain is to be performed for diagnostic purposes. The report by Paueksakon et al [1] represents an excellent first step in validation of an antibody for amyloidosis. Moving forward by adjusting the conditions one variable at a time until a balance is achieved, such that
0046-8177/© 2014 Elsevier Inc. All rights reserved.
LECT2 amyloidosis cases are positive and the non-LECT2 are negative, will likely result in a reliable assay. The antigen retrieval step is frequently problematic for immunohistochemical assays for amyloid subtyping, as overretrieval results in a false-positive staining pattern similar to that demonstrated in the photomicrographs from Paueksakon et al [1]. Most amyloid subtypes require little-to-no antigen retrieval to elicit a diagnostic reaction [3]. In all likelihood, adjusting this step will increase the specificity of the stain without sacrificing sensitivity. Thank you. Christopher Larsen MD Nephropath Little Rock, AR 72211 E-mail address: [email protected] http://dx.doi.org/10.1016/j.humpath.2014.06.024
References [1] Paueksakon P, Fogo AB, Sethi S. Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining. HUM PATHOL 45:1445-50. [2] Fitzgibbons PL, Bradley LA, Fatheree LA, et al. Principles of analytic validation of immunohistochemical assays: guideline from the College of American Pathologists pathology and laboratory quality center. Arch Pathol Lab Med. Published online ahead of print 2014 Mar 19. [3] Kebbel A, Röcken C. Immunohistochemical classification of amyloid in surgical pathology revisited. Am J Surg Pathol 2006;30:673-83.
Leukocyte chemotactic factor 2 amyloidosis can be reliably diagnosed by immunohistochemical staining—reply The authors reply: We thank Dr Larsen for his interest in our recent publication [1]. He noted that in his experience, LECT2
2180 immunohistochemical staining is a highly sensitive and specific test for LECT2 amyloidosis, which is in contrast to our study. We agree with Dr. Larsen’s comments about the general immunohistochemical principle that the aggressive antigen retrieval step may result in false positive staining. However, the LECT2 antibody that we utilized in this study was carefully validated as optimized for our purposes. It is noteworthy that only 4 of 45 cases of amyloid light-chain and amyloid A amyloidosis were LECT2 positive, which would be very unusual in a true example of over-retrieval. While further studies are needed to confirm our initial findings (as correctly pointed out by Dr. Larsen, this will require the analysis of a larger cohort of LECT2 amyloidosis from other, more common forms of renal amyloidosis), our studies suggest that the immunohistochemical diagnosis of LECT2 amyloidosis may need to be confirmed by laser microdissection with mass spectrometry (LMD/MS) analysis of renal tissues when the staining pattern shows equivocal intensity.
Correspondence Paisit Paueksakon MD⁎ Agnes B. Fogo MD Department of Pathology, Microbiology, and Immunology Vanderbilt University Medical Center Nashville, TN 37027 ⁎Corresponding author E-mail address:[email protected] Sanjeev Sethi MD Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, MN 55905 http://dx.doi.org/10.1016/j.humpath.2014.06.026
Reference [1] Paueksakon P, Fogo AB, Sethi S. Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining. HUM PATHOL 2014;45:1445-50.
