AL-amyloidosis is underdiagnosed in renal biopsies

Lea Novak1, William J. Cook1, Guillermo A. Herrera4,5,6 and Paul W. Sanders2,3,7

Departments of 1 Pathology, 2 Medicine, and 3 Physiology and Biophysics, University of Alabama at Birmingham, AL, Departments of 4 Pathology, 5 Medicine, and 6 Cellular Biology and Anatomy, LSU Health Sciences Center, Shreveport, LA and 7 Veterans Affairs Medical Center, Birmingham, AL, USA

Correspondence and offprint requests to: Paul W. Sanders, MD, Division of Nephrology, LHRB 642, University of Alabama at Birmingham, 1530 Third Avenue, South Birmingham, AL 35294-0007, USA. Email: psanders{at}uab.edu



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Renal amyloidosis is associated with a variety of underlying disease processes. Although amyloid is identical in appearance in these diseases, the precursor proteins are different. Immunofluorescence microscopy has been used as the primary tool in the diagnostic evaluation of the underlying cause of renal AL-amyloidosis. The purpose of this study was to document the sensitivity of immunofluorescence microscopy in AL-amyloidosis.

Methods. We reviewed 36 renal biopsies from patients with amyloidosis collected in two medical centres. All biopsies showed characteristic fibrillary deposits of amyloid on electron microscopy and stained positive with Congo red or Thioflavin-T.

Results. Among these 36 patients, immunofluorescence staining for {lambda} and {kappa} light chains was negative or equivocal in 14 biopsies. Of these 14 patients, two patients had evidence of AA-amyloidosis. Twelve patients were found subsequently to have a plasma cell dyscrasia or multiple myeloma with monoclonal immunoglobulin and/or free light chains on immunofixation electrophoresis of urine or serum, and with evaluation of the bone marrow. Thus, 12 of 34 patients (35.3%) with proven AL-amyloidosis had negative immunofluorescence staining for {kappa} and {lambda} light chains.

Conclusions. The data demonstrated the low sensitivity of immunofluorescence microscopy in the detection of AL-amyloidosis in the kidney and underscore the need to pursue additional diagnostic studies to identify this problem.

Keywords: amyloidosis; kidney biopsy; multiple myeloma; plasma cell dyscrasia; renal biopsy



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Amyloidosis is a generic term for a heterogeneous group of diseases that have in common tissue deposits of extracellular fibrillary proteins of similar structure but different chemical composition [1,2]. In primary amyloidosis, the amyloid fibril, designated AL, is derived from immunoglobulin {kappa} or {lambda} light chains or their fragments [3–7]. The second major form of amyloid occurs in the setting of chronic inflammation or familial Mediterranean fever and is derived from serum amyloid A, an acute phase reactant protein; the fibril type is designated AA [8]. One of the three patients with ‘Bright's disease’ originally described by Richard Bright in the 19th century had amyloidosis, presumably AA since she had cavitary pulmonary tuberculosis [9]. Immunoglobulin heavy chains have been shown to polymerize to form amyloid, termed AH [10]. Other types of amyloidosis are hereditary and related to mutations in transthyretin [11], apolipoprotein A-I [12], lysozyme [13] or fibrinogen A {alpha}-chain [14].

The diagnosis of amyloidosis rests with histological and ultrastructural demonstration of deposition of amyloid. Because the kidney is commonly affected in amyloidosis, renal biopsy is frequently the diagnostic test. The deposits may be present in the mesangium, glomerular capillary walls, interstitium or renal vessels. Amyloid appears homogeneous, acellular and amorphous on haematoxylin–eosin-stained sections. In advanced disease, glomeruli that are replaced by amyloid are hypocellular and may resemble globally sclerotic glomeruli. The gold standard for the diagnosis of all types of amyloid is the Congo red stain, which exhibits green birefringence when viewed under polarized light, or Thioflavin-T. On electron microscopy, amyloid is characterized by non-branching, randomly oriented fibrils measuring 9–11 nm in thickness. These findings are identical with all types of amyloid proteins, regardless of the precursor protein, and therefore provide no insight into the underlying pathogenesis of the amyloidosis.

Determination of the composition of the amyloid has traditionally been made using immunohistochemical techniques. Fluorescein-labelled antibodies against {lambda} and {kappa} light chains are routinely used to identify AL-amyloid in renal biopsies, but our experience suggested that the sensitivity of this approach was low. The purpose of the present study was to determine the reliability of the {kappa} and {lambda} immunofluorescence stains in the diagnosis of renal involvement from AL-amyloidosis.



   Subjects and methods
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
The Institutional Review Board of the University of Alabama at Birmingham approved the project. From January 1996 to June 2000, 3882 kidney biopsies were collected at the University of Alabama at Birmingham, Birmingham, AL, and LSU Health Sciences Center, Shreveport, LA. Of these, amyloidosis was found in 36 renal biopsies (0.9%). Among those cases, 14 biopsies were reported negative for routine immunofluorescence staining for {lambda} and {kappa} light chains, while the remainder showed immunoreactivity for either {lambda} or {kappa} light chains in the glomerulus. All showed fibrillary deposits on electron microscopy diagnostic of amyloid and stained positive with either Congo red or Thioflavin-T. These patients were included in this study. Patients' medical records were reviewed and results from serum/urine immunofixation electrophoresis and bone marrow biopsy with flow cytometry were documented, when available. Surgical pathology reports and electron micrographs were reviewed along with histological slides stained with haematoxylin–eosin, periodic acid–Schiff, silver methenamine and trichrome. Remnants of frozen renal biopsies were retrieved for confirmatory immunofluorescence staining for {kappa} and {lambda} light chains.

All renal biopsies were tested for the presence of IgG, IgA and IgM, as well as C1q, C3 and albumin, by immunofluorescence staining. Each biopsy was also tested with antibodies against {kappa} and {lambda} free light chains from two different companies, as well as antibodies against AA-amyloid. In the original biopsies, fresh frozen renal tissue was sectioned at 3 µm thickness and air-dried. Sections were washed in phosphate-buffered saline (PBS) for 5 min and then stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-human {kappa} and {lambda} light chain antibodies (ICN/Cappel, Aurora, OH), 1:100 dilution in 5% bovine serum albumin (BSA). For confirmation in this study, biopsy sections were fixed for 20 min in 3.7% paraformaldehyde/PBS at room temperature, and then washed in PBS three times. After blocking for 30 min in 5% BSA at room temperature, the sections were incubated with rabbit anti-human {kappa} or {lambda} light chain (DAKO, Carpinteria, CA), 1:100 dilution in 5% BSA, for 2 h at room temperature and washed in PBS three times. The sections were incubated with tetramethylrhodamine isothiocyanate conjugated anti-rabbit IgG (Roche Diagnostic Corp., Indianapolis, IN), 1:100 dilution in 5% BSA, for 1 h at room temperature and washed in PBS three times. The sections were cover-slipped using aqueous mounting medium. Control slides containing renal biopsies of the patients with amyloidosis that were previously positive for {kappa} and {lambda} light chains were stained in parallel with renal biopsies included in this study. The sections were also stained for AA-amyloid using mouse antibody directed against human amyloid A component (DAKO), 1:200 dilution in 5% BSA, followed by FITC-conjugated anti-mouse IgG (Roche Diagnostic Corp.). Controls using specimens obtained from patients with documented AA-amyloid were also examined. The slides were examined in routine fashion using an immunofluorescence microscope.



   Results
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 Subjects and methods
 Results
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 References
 
Among the 36 patients with biopsy-proven renal amyloidosis identified over this time period, 14 showed negative immunofluorescence staining for {kappa} and {lambda} light chains (38.9%) and were included in this study (Table 1). Thirteen patients were Caucasians and one was African-American. Six were females and eight were males; the ages ranged from 42 to 74 years. The diagnosis of amyloidosis was unexpected in the majority of these patients prior to renal biopsy. Light microscopy revealed deposition of eosinophilic material suggestive of amyloid in glomeruli and vessels. All patients had positive staining with Congo red or Thioflavin-T. Ultrastructural evidence of tissue deposits of extracellular, randomly branched fibrils diagnostic of amyloid was observed in every sample. Immunofluorescence microscopy was performed using two different commercially available antibodies and procedures and was completely negative for {kappa} and {lambda} light chains. Immunofluorescence using antibodies directed against IgG and IgA was also completely negative; stains for IgM, C1q and C3 were also negative or non-specific. One patient had positive immunohistochemical staining for AA-type amyloid; the other patient had insufficient biopsy tissue remaining to perform additional staining for AA-amyloid, but he had negative serological evaluation for a plasma cell dyscrasia and a clinical history of T5 paraplegia complicated with sacral decubiti and chronic osteomyelitis.


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Table 1. Summary of findings of the fourteen patients in the study

 
Analysis of the serum or urine identified monoclonal free {kappa} or {lambda} light chains in eight patients. {lambda} light chains predominated, with only one patient testing positive for {kappa} light chains. Immunofixation electrophoresis also identified monoclonal IgG-{lambda} in three patients and monoclonal IgA-{lambda} in one patient. Immunofixation electrophoresis did not detect an abnormality in two patients (nos 6 and 14), both of whom had clinical or immunofluorescence microscopic findings compatible with AA-amyloidosis. The results were unknown in two other patients (nos 8 and 10), although both patients had a bone marrow biopsy that identified a plasma cell dyscrasia. Bone marrow biopsy showed occasional multinucleated plasma cells and increased numbers of {lambda}-staining cells in patient no. 8, although flow cytometry failed to show a monoclonal population of plasma cells. Bone marrow evaluation showed increased numbers of plasma cells in two patients and findings consistent with multiple myeloma in seven patients, according to standard criteria [15]. In three patients, the biopsy did not demonstrate a plasma cell dyscrasia. Two of these patients had evidence of AA-amyloidosis and the other patient (no. 2) had other evidence of a monoclonal gammopathy by immunofixation electrophoresis. Two patients (nos 4 and 9) had monoclonal free {lambda} light chains detected with immunofixation electrophoresis and did not have bone marrow biopsy; patient no. 9 was treated with chemotherapy without bone marrow biopsy.

In summary, of these 14 patients, 12 were found to have a plasma cell dyscrasia confirmed by at least one of the following tests: urine and serum immunofixation electrophoresis, flow cytometry of bone marrow aspirates, or histological examination of bone marrow biopsy. Two of the 14 patients had evidence of a chronic inflammatory process and were diagnosed clinically as AA (secondary) amyloidosis.



   Discussion
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
The present study is important to clinicians caring for patients with amyloidosis, because treatment of AL-amyloidosis differs from treatment for other types of amyloidosis. While immunofluorescence is commonly used to document deposition of immunoglobulin proteins in the kidney, the present study demonstrated the low sensitivity of this procedure in patients with renal involvement from AL-amyloidosis. In 1986, Gallo and associates were able to use immunofluorescence to classify 88% of patients with renal amyloidosis. Two patients in that study had negative staining for light chains and AA protein, but biochemical analysis demonstrated that the amyloid was composed of {lambda} light chains [16]. Using immunohistochemistry, a more recent study of 316 patients could confirm the composition of AL-amyloid from various tissues in only 38% [17]. In the present study, despite the use of highly sensitive immunofluorescence staining of kidney tissue, glomerular deposition of light chains was not detected in 12 of 34 (35.3%) patients with documented renal involvement from AL-amyloidosis. Aside from the two patients with AA-amyloidosis, using other tests, a plasma cell dyscrasia was confirmed in every patient in the present study. Thus, the false-negative rate of immunofluorescence staining for glomerular light chain deposition in AL-amyloidosis was 35.3%.

There are several potential explanations for negative immunofluorescence for light chains. Technical issues were ruled out since the control slides were reactive and two different commercially available antibodies were used. The amount of amyloid deposited in the kidney might also account for the false negativity, but all patients in this study had deposits of sufficient size to be seen ultrastructurally. Although renal biopsy was performed early in the course of the disease in some patients, other patients with negative immunofluorescence staining had abundant deposits and some patients with positive immunofluorescence staining had small deposits, so this explanation seemed unlikely. AL-amyloid has been shown to consist of the variable domain of immunoglobulin {kappa} or {lambda} light chains as well as intact light chains [3–7]. Because of sequence variability of this domain and alterations in the structure of the pathological light chains in the amyloid, antibodies that have been raised against intact light chains might not react to an amyloid fibril, but could be used to detect circulating intact light chains. The process of amyloid fibril formation also often fragments the light chain, further preventing detection by commercially available antibodies. This is the probable explanation of our findings.

As was the case in this study, amyloid is far more often associated with {lambda} light chains. A striking tropism of the {lambda}VI variant for the kidney has been demonstrated [18]. Immunoglobulin heavy chains can also polymerize to form AH-type amyloid [10], although no patient in the present study had positive immunofluorescence staining for heavy chains in the kidney and a majority of the patients (75%) with a final diagnosis of AL-amyloidosis had evidence of monoclonal light chains in the circulation, urine or bone marrow. Categorizing the type of amyloid through the use of antibodies that either detect specific subclasses of {kappa} and {lambda} light chains or are directed against purified {kappa} and {lambda} amyloid fibrils has not been documented but may be another avenue to pursue in situations where immunofluorescence staining using polyclonal anti-{kappa} and anti-{lambda} antibodies is negative.

AA-amyloidosis was present in two patients who had clinical evidence of a chronic inflammatory process, along with unremarkable immunofixation electrophoresis of serum and urine, and AA staining of the glomeruli (one patient).

Perhaps the most difficult diagnosis to exclude is hereditary amyloidosis, a very rare autosomal dominant disease related to mutation of transthyretin [11], apolipoprotein A-I [12], lysozyme [13] or fibrinogen A {alpha}-chain [14]. In one study of 350 patients who had presumed AL-amyloidosis, 36 patients were observed to have mutations of one of these proteins, usually fibrinogen A {alpha}-chain or transthyretin [17]. While the predominant clinical features of the majority of these patients were related to cardiomyopathy or neuropathy, nephropathy can occur [11–14]. Although none of the patients in the present study had a known family history of renal disease or amyloidosis, many patients in the study by Lachmann and associates also lacked a relevant family history [17]. Other references, however, support the importance of the family history in diagnosing hereditary amyloidosis [11–14]. Low levels of intact monoclonal immunoglobulin light chains were detected in 24% of patients with hereditary amyloidosis, but none had monoclonal free light chains in the urine [17]. In the present study, the diagnosis of a plasma cell dyscrasia was confirmed using additional tests. Patient no. 2 had a monoclonal IgG-{lambda} gammopathy and an unremarkable bone marrow evaluation, but also had suppression of serum levels of IgM and IgA and elevated serum ß2-microglobulin concentration and was therefore considered to have multiple myeloma. Although not used in this study, Lachman et al. demonstrated circulating monoclonal light chains in the majority of patients with AL-amyloidosis using a sensitive nephelometric immunoassay [19].

In conclusion, patients with negative immunofluorescence microscopy represent a challenging diagnostic dilemma for the clinician and warrant careful examination to confirm the type of amyloidosis. While immunofluorescence microscopy of renal tissue is an important diagnostic tool, currently available antibodies to {kappa} and {lambda} light chains do not reliably detect light chains in AL-amyloid deposits in the kidney; therefore, negative light chain staining in association with amyloid does not rule out AL-amyloidosis. All patients with renal amyloidosis merit careful documentation of the family history and a complete evaluation for the presence of a plasma cell dyscrasia. Because of the potential confusion with hereditary amyloidosis and obvious therapeutic implications, immunofixation electrophoresis of both serum and urine or other sensitive immunoassays that accurately quantify serum levels of {kappa} and {lambda} light chains [19] should be carried out to identify the presence of monoclonal free light chains in the serum or urine, along with bone marrow evaluation with attendant flow cytometric assay of the cellular content of the marrow. Finally, although potentially costly if all amyloid precursors are examined, if confusion persists, a genetic cause for the amyloidosis should be pursued.



   Acknowledgments
 
This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, by a grant from the Multiple Myeloma Research Foundation, and by generous donations from friends and family of Nila and Larry Minor. A portion of this work was presented at the annual meeting of the College of American Pathologists in Washington DC in 2001 and was published in abstract form (Mod Pathol 14:189A, 2001; and Lab Invest 81:189A, 2001).

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 6.04
Accepted in revised form: 31. 8.04