The amyloidoses are a group of diseases characterized by deposition of proteinaceous fibrils with a cross–β-sheet molecular structure. This structure is responsible for the binding affinity of Congo red dye and the green birefringence observed with polarized light (1). Extracellular deposition of amyloid fibrils results in loss of organ function. Deposition of amyloid can be localized (restricted to 1 organ or site of the body) or systemic (in various organs and tissues throughout the body). Systemic amyloidosis is a disease with high mortality because of the progressive and widespread deposition of amyloid in vital organs (2).
Amyloid deposition in the kidneys, nerves, spleen, vitreous body, and abdominal fat is seen exclusively in patients with the systemic forms of amyloidosis. The detection of amyloid in the bone marrow, heart, liver, gastrointestinal tract, lung, or joint nearly always indicates systemic amyloidosis. The 3 major types of systemic amyloidosis are AA, AL, and ATTR amyloidosis. These types can be distinguished by the nature of the precursor protein of the fibrils. The underlying diseases require different treatment (2). In all of the systemic amyloidoses, accurate and early diagnosis is extremely important for clinical management. Early effective treatment will retard or even stop further deposition of amyloid, but the potential effect of treatment will be dependent on the extent of disease progression at the time of diagnosis.
The diagnosis of amyloidosis is based on histologic analysis. A biopsy specimen should demonstrate positive staining with Congo red dye and characteristic apple-green birefringence observed with polarized light. Biopsy of an involved organ is the diagnostic gold standard. However, biopsy of a clinically suspected site (kidney, liver, or heart) is an invasive procedure, and significant complications, such as bleeding, are a potential risk. Both biopsy of the rectum and abdominal fat aspiration are frequently used to detect systemic amyloidosis in patients with signs or symptoms of the disease.
Since its introduction in the 1960s, biopsy of the rectum has been considered by many investigators to be the gold standard for screening, with a sensitivity of ∼80% (3–6), but this method is rather inconvenient and time-consuming. Aspiration of abdominal fat tissue is a simple, safe, and less inconvenient method, which was originally described in 1973 (7). It is a fast bedside or outpatient procedure (8). As previously mentioned, amyloid deposition in abdominal fat tissue is seen exclusively in the setting of systemic amyloidosis. The specificity of this test, provided that the staining procedure is performed correctly, approaches 100% (9–12). Sensitivity values vary greatly, from 52% to 88% (5, 9–18). This wide range might be attributable to the amount of fat tissue, differences in experience with the staining and scoring of biopsy specimens, characteristics of the patients, the size of the study group, and the type of systemic amyloidosis.
Because of the wide range of sensitivity reported in the literature, the clinical utility of abdominal fat aspiration for detecting systemic amyloidosis remains to be determined. In clinical practice, other more invasive biopsies often are performed in order to establish the diagnosis. In the present study, we analyzed our experience with abdominal fat aspiration over a 10-year period. The aim of the study was to assess the diagnostic accuracy of abdominal fat aspiration and the clinical utility of this method.
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- PATIENTS AND METHODS
Accurate and early diagnosis of systemic amyloidosis is essential in the clinical management of the disease. Fat tissue analysis is the best detection method and is associated with high clinical utility.
Amyloidosis is a histologic diagnosis, and it would be advantageous to have a specific test that does not require biopsy of a major organ (such as the kidney, liver, or heart), because of the risk of complications associated with such procedures. Even biopsy of the rectum, which for a long time has been considered the gold standard for screening, is an inconvenient, time-consuming, expensive, and risky procedure compared with fat tissue aspiration. Although in our study the groups were small and subjects in the control group were relatively young, we confirmed the specificity of thoroughly examined fat tissue to be 100% (95% CI 92–100%). The utility of this test is based on its excellent positive predictive value, i.e., that fat tissue positivity actually proves the presence of systemic amyloidosis. Despite these excellent values, one should keep in mind that high specificity depends on correct application of standard protocols using the Congo red staining method described by Puchtler et al (22–24). In clinical practice, it is good to recognize that false-positive results might occur. First, pale-yellow birefringent fibrin, collagen, or elastin fibers should be distinguished from the typical apple-green birefringence of amyloid. Second, exogenous polysaccharide materials, such as plant cell walls, starch, cotton fibers, and various fungi, stain with Congo red dye and show green birefringence (23, 24).
According to the literature, the sensitivity of fat tissue for diagnosing systemic amyloidosis is still undetermined, because of the wide range in the values for sensitivity (52–88%) (14, 15). Therefore, in routine clinical practice, invasive biopsies of visceral organs are still performed in many cases. Our study shows that in routine clinical practice, the sensitivity of subcutaneous fat tissue analysis is 80–85%, provided that an adequate amount of fat tissue (at least 30 mg) is properly processed and examined, and provided that 3 glass slides per patient are examined instead of only 1. Systematic use of rectal, stomach, bone marrow, and especially abdominal fat tissue, as practiced in the screening group in this study, results in a diminished need to perform biopsies of major organs compared with the need in the standard group, as illustrated for the kidneys, for which the necessity to perform biopsy decreased ∼30% (from 39% to 10%).
Thorough assessment of fat smears stained with Congo red dye increases sensitivity to >90% in AA and AL amyloidosis, without decreasing specificity. This is probably attributable to the extent of experience in assessing fat tissue, as discussed by Linke et al (25). These values are sufficiently high to propagate a more prominent role in clinical practice for fat tissue analysis in the detection of systemic amyloidosis. In patients with FMF, the sensitivity of fat tissue analysis seems to be lower than that in patients with other inflammatory diseases (26). Therefore, the high sensitivity of fat aspiration for AA amyloidosis in this study may be partly explained by the low number of patients with FMF.
Pitfalls in the analysis of fat tissue are false-negative results due to an insufficient amount of material (too little fat tissue aspirated), inadequate staining technique, improper use of polarizing instruments, and insufficient light intensity. Therefore, in case of negative findings in the fat aspirate from a patient with a persistently high clinical suspicion of amyloidosis or progressive disease for which there is no other explanation, fat aspiration should be repeated, and the aspirate should be sent for further analysis to a center with experience in this procedure. Recently developed techniques such as (immuno)chemical analysis of fat tissue may facilitate automated detection of amyloid in fat tissue (21, 27, 28). The development of generally available techniques for automated detection of amyloid in fat tissue with high accuracy would be a major step forward, because amyloid detection would then become independent of the experience and dedication of the observer.
The sensitivity of fat tissue aspiration turns out to be at least as good as (and, in the case of thorough examination, even better than) the sensitivity of biopsy of the rectum. Notably, if the findings in fat tissue are negative, the profitability of subsequent biopsy of the rectum is negligible. Therefore, if findings in a thoroughly examined fat smear are negative and clinical suspicion remains high, the next step is a directed biopsy of an organ suspected to be involved. As expected, the sensitivity of bone marrow biopsy turned out to be rather low (63%), whereas the sensitivity of biopsy of the kidney, liver, and heart was high (87–98%), as shown in Figure 1 (5). The 63% sensitivity associated with bone marrow biopsy does signify that when detection of an M protein suggests the need for a bone marrow biopsy in order to look for plasma cell dyscrasia, additional staining with Congo red dye may help to detect approximately two-thirds of the patients with amyloidosis, with minimal additional effort.
The utility of fat aspiration in clinical practice is high, as shown in the screening group, because the method easily identifies a group of currently treatable disorders. As reported above, the results of fat tissue aspiration were positive in 66 (41%) of the 162 patients. In 13 (20%) of these patients, fat tissue was the only tissue positive for amyloid. Only 3 (3%) of 93 patients with negative results of fat tissue analysis had amyloid in another tissue. Therefore, similar to the results in the standard group, the sensitivity of fat tissue aspiration in the screening group was higher than the sensitivity of biopsy of the rectum and bone marrow biopsy.
The high accuracy of fat tissue aspiration, along with the other advantages of being inexpensive, simple, fast, safe, and more convenient for the patient, makes fat aspiration the preferred method to use when searching for amyloid. Early diagnosis may have a favorable effect on clinical outcome, whereas both patient and clinician benefit from the reduction of uncertainty. Currently, the only disadvantage of fat tissue aspiration is its unsuitability for using immunohistologic analysis to establish the type of amyloid involved. This disadvantage can be overcome by using other approaches, such as biochemistry, immunochemistry, and immunoelectron microscopy (21, 27–29). However, these other techniques are not yet generally available. In most cases, typing of amyloid is not strictly necessary, because the clinical data indicate the type of amyloid. In the rare cases in which typing is obligatory, our current policy is to perform another biopsy (generally of the rectum) that permits immunohistologic characterization.
As Andrews et al already reported, “the yield of a subcutaneous fat aspirate in patients with isolated peripheral neuropathy and no other associated family history, signs, or symptoms of amyloidosis is low” (30). The results of the current study confirm this observation, because amyloid was detected in none of the 20 patients for whom there was no suspicion of a specific type of systemic amyloidosis (no chronic underlying disease, no plasma cell dyscrasia, no positive family history). In most of these patients, the indication for fat aspiration was neuropathy.
In the screening group, the percentage of subjects for whom proteinuria was an indication for biopsy was higher in the subgroup with AL amyloidosis than in the subgroups without AL amyloidosis. In contrast, among patients with chronic (rheumatic) disease, proteinuria was not strongly indicative of AA amyloidosis.
In conclusion, the results of our study show that subcutaneous fat aspiration is the method of choice for routine screening for systemic amyloidosis, because it is a simple, convenient, inexpensive, fast, safe, bedside procedure with high diagnostic accuracy. If the results for properly examined fat tissue are positive, the diagnosis is established, and there is no need for another invasive biopsy. If the results of fat tissue aspiration are negative and the clinical suspicion of systemic amyloidosis remains high, unstained fat tissue may be sent to a tertiary center for thorough evaluation. If results are still negative, the additional value of subsequent biopsy of the rectum or bone marrow biopsy is low.