Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.
Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.
Introduction
Systemic amyloidosis is a highly variable disease that refers to extracellular deposition of various proteins that are collectively referred to as amyloid. Deposition of these amyloid fibrils with beta pleated configuration leads to various clinical presentations depending on the organs involved. The most clinically relevant manifestations of systemic amyloidosis are noted when there is involvement of critical organs such as the heart, liver, and/or kidney. Historically, these involved organs would be biopsied to demonstrate the presence of amyloid. These moderately invasive procedures carried a significant risk including hemorrhage. Fat pad aspiration has since been shown to provide a reliable and noninvasive method for the detection of amyloid in systemic amyloidosis 1. This procedure is essentially comparable to the liposuction of the subcutaneous fat in the anterior abdominal wall under local anesthesia to retrieve fibroadipose tissue for evaluating scant amyloid deposits in small blood vessel walls 2.
The most frequently utilized method for amyloid identification in tissue remains the characteristic apple-green birefringence pattern seen when Congo red stained sections are visualized under a polarized light microscope 3. When fat pad aspiration is performed, Congo red stains can be done on either direct smeared slides or cell block preparations of the aspirated adipose tissue. However, patients in early stages of amyloidosis have scant amyloid deposits, which greatly reduces the sensitivity of Congo red stained cell block sections 4,5. Ultrastructural evaluation of fat pad aspirates by electron microscopy has better reproducibility and improved sensitivity 4. Therefore, it is recommended to submit all fat pad aspirates for both preparation of a cell block and for performing electron microscopy 2.
Fat pad aspiration is a relatively low cost and noninvasive method for obtaining tissue to diagnose systemic amyloidosis. This article describes fat pad aspiration procedure along with details about sample processing to submit specimen for both Congo red staining and ultrastructural evaluation by electron microscopy. In this video, we demonstrate this reproducible and simple procedure to retrieve optimal diagnostic material.
1. Performing the FNA of Anterior Fat Pad
A. Anesthetization of the local area (Figure 1 & 2)
B. Performing fat pad aspiration (Figure 3)
(Application of local anesthesia prior to performance of fat pad aspiration may be bypassed, depending on the regional and individual preferences. Properly performed FNAB procedure for anterior fat pad aspiration can be completed in one prick. However, depending on the pain threshold of individual patient, maneuvering of 18G needle back and forth in to the subcutaneous fat tissue is relatively distressing. The anesthetization method described here achieves the effect in only two pricks with 25G needle and averts the pain with improved tolerance to the procedure.)
2. Specimen Processing
Figure 1. Area to be anesthetized for FNAB of anterior fat pad.
Figure 2. Anesthetization of the local area.
Figure 3. Performing FNAB of anterior fat pad.
Figure 4. Processing of anterior fat pad aspirate to be submitted to laboratory for detecting amyloid deposits.
3. Representative Results
Figure 5. Amyloid in the wall of small blood vessels in fibroadipose tissue fragments. Fibroadipose tissue was formalin-processed, paraffin embedded, stained with Congo red, and examined by polarizing light microscopy. Amyloid in the wall of small blood vessels shows apple green birefringence (white arrow).
Figure 6. Tissue lacking amyloidosis. The apple green birefringence is absent in tissues of a different patient without amyloidosis. Blue birefringence (white arrow) of collagen fibers, are usually present in almost all specimens.
Figure 7. Fibrils consistent with amyloid in the blood vessel wall. Electron micrograph of straight, non-branching, randomly-scattered 8-10 nm diameter fibrils formed by amyloid in the blood vessel wall.
Diagnosis of amyloidosis is usually achieved with a tissue biopsy of the affected organs such as the kidney, liver, and/or heart. This approach has high diagnostic yield, however, is invasive and may be associated with complications including hemorrhage 2. Rectal, gingival, and bone marrow biopsies were preferred for diagnosis as relatively less invasive approach in 1960s 6,7,8. Abdominal fat pad aspiration was reported in 1973 as a safe, minimally invasive, simple, and less expensive procedure for the tissue diagnosis of systemic amyloidosis 1. However, the details of the procedure and approach to processing of the retrieved specimen is not coherently reported. This video and the article describe the procedure step by step.
The approach for detecting amyloid in fat pad aspiration, similarly, is also not well standardized with a few studies comparing and evaluating various approaches 1,5,6,7,8. Evaluation of anterior fat pad aspirates with Congo red staining is less sensitive with lower interobserver reproducibility especially in early cases of amyloidosis with scant amyloid deposits 5. Immunohistochemistry performed on formalin fixed paraffin-embedded cell block sections and Congo red fluorescence performed on cytology smears have been reported to improve the diagnostic sensitivity 9,10. Electron microscopy improves the detection and identification of scant amyloid fibrils in small blood vessel walls in the fibroadipose tissue in fat pad aspirates 4, 5. Based on this information, this article describes processing of the specimen to prepare cell blocks (for evaluation of Congo red stained cell block sections under polarizing microscope for diagnostic apple green birefringence and also for immunohistochemistry as indicated) and for electron microscopy. However, depending on the approach selected for evaluation of the specimen for amyloid, it may be subjected to cytology smear preparation, cell block preparation, and processing for electron microscopy. Some laboratories perform the tests on the smears prepared from the aspirated fibroadipose tissue fragments and stained with Congo red to study the fluorescence 10.
Generally in a late amyloidosis, the light microscopy with Congo red polarizing microscopy may be sufficient; however, in early stages of the disease Congo red staining with polarizing microscopy on cell block sections is less sensitive and reliable for fat pad aspirates 4,5,11. Other special stains including Thioflavin T may be used with similar limitations. Other approaches to detect amyloid include evaluation of the aspirate smears for amyloid with Congo red polarizing microscopy and fluorescence 10. In our experience this method is less reproducible. Evaluation of the blood vessels in fat pad for amyloid by electron microscopy overcomes these limitations 4,11. Further characterization of amyloid has been reported by immunoelectron microscopy 12; however, these methods may not be available widely in a non-tertiary care setting.
Needles used for performing aspiration procedures may be categorized according to their gauge sizes into fine (21-25G), intermediate (18-20G), and large (eg- 14G) 11. Anterior fat pad aspirations have been reported to be performed by using variable gauges ranging from intermediate (18 to 20G) to fine (21 to 22G) 2,10. Most of the mass lesions are aspirated with finer gauge needles (such as 25G) to obtain good cytology smears along with additional passes with wider gauge needle (such as 18G) to retrieve additional sample for cell blocks.
During anterior fat pad aspiration, the cohesive fibroadipose tissue does not aspirate well with finer needles. Cell block preparation and electron microscopy are important for evaluation of small blood vessel walls in fibroadipose tissue fragments in the fat aspirates. Wider gauge needles (such as 18G) are recommended to yield diagnostic adequate material 2,10. As the procedure is comparable to the conventional FNAB, fat pad aspiration is referred to as FNAB even though wider gauge needles may be used for performing it 5.
The authors have nothing to disclose.
We thank Ms. Bonnie Phetteplace, RN for assistance during video graphing the FNAB procedure demonstration.
Alcohol swabs, gauze pads, marking pen, 10 mL syringes x2, 1% lidocaine (local anesthetic- If lidocaine is used alone, it causes an initial burning sensation after instillation. This can be prevented by using a 1:1 mixture of 1% lidocaine and 1% sodium-bi-carbonate), 18 gauge(G) 1 ½ inch needles x2 (for performing FNAB), 25G 1 ½ inch needle (for injecting local anesthetic), FNA syringe holder (“gun”), bandage, vial of glutaraldehyde (for electron microscopy), and biopsy container of 10% formalin.