Fat Tissue Analysis in the Management of Patients with Systemic Amyloidosis

Abstract

Aspiration of abdominal subcutaneous fat tissue is a safe, inexpensive, reliable, minimally invasive, and easy procedure with high diagnostic yield that can be done early after clinical suspicion of amyloidosis and can be repeated if required anytime during the course of the disease. Material obtained in this way is highly suitable for detection, typing, and quantification of amyloid and can also be used for chemical tissue analysis using proteomic techniques. The procedure has great potential to enable dynamic research of local tissue factors involved both in deposition and removal of amyloid in vivo. In the Appendix of this chapter, we describe the current practice of this procedure in our center.

Appendices

Appendix: Fat Aspiration Technique and Tissue Analysis Currently Practiced in Groningen

Fat Aspiration Technique

Stepwise Description of the Procedure

Aspiration of abdominal subcutaneous fat tissue is a simple outpatient procedure and a modification of the procedure was described by Gertz [17]. It should be noted that it takes at least 10–15 min to avoid unnecessary pain and bruising and to get adequate material. The patient should be told that bruising might occur. For a description and instruction video of the fat aspiration procedure one can visit our website www.amyloid.nl [103]. Alternative procedures have been described, such as by Kettwich et al. [104] and also for preparing and performing electron microscopy on fat tissue [105, 106].

See Table 16.3 for the equipment we use. A syringe of 10 ml is connected by a valve system to a 16-gauge needle (Fig. 16.5a). After closing the valve, the plunger is pulled out, fixed transiently between squeezed thumb and finger, the cap of the lidocaine needle is reused elegantly by positioning it upside-down inside the plunger (Tarek’s trick) to fix firmly and definitely the position of the plunger, and thus maintaining negative pressure in the syringe during aspiration (Fig. 16.5b). The skin of the patient is marked and cleansed (e.g., with chloorhexidine) at both sides of the umbilicus at about 7–10 cm distance. Check first that the patient is not allergic to lidocaine. Skin and subcutaneous tissue (three directions, see below) are then anesthetized with lidocaine (each side 2 ml = 20 mg).

Table 16.3 Equipment for the fat aspiration

Fig. 16.5

(a) The closed valve; reusing the needle cap. (b) Pull and fix the plunger and position the needle cap

After inserting the needle beneath the skin, the valve is opened to start aspiration of fat tissue (Fig. 16.6a). The needle can be moved into three directions (Northeast, East, and Southeast) at the left side of the abdomen and mirror-wise at the right side. The aspiration procedure should be performed slowly and gently into each of the three directions, going to and fro with some axial rotation, and one should realize that it will take some time before the needle will be filled with fat tissue and the first fat can be seen passing the valve and entering the top of the syringe. This should be continued at both sides of the umbilicus until at least 60 mg of fat tissue has been collected (Fig. 16.6b). After the procedure has been finished, the puncture site should be covered with a band aid and pressed for a while to prevent substantial bruising.

Fig. 16.6

(a) Insert the needle beneath the skin and open the valve. (b) Yield: about 60 mg of fat tissue

The next step may be as simple as this: Seal the syringe, keep it cool (4 °C) until shipment, and send it at room temperature to a diagnostic center (e.g., UMC Groningen) for analysis as soon as possible, but at the latest arriving there within 1 week after the procedure.

Frequently Encountered Problems During the Procedure

Two technical problems can be encountered during aspiration: no tissue at all or too much blood entering the syringe.

If no fat appears in the syringe or the aspiration has stopped completely for some time, the needle may have become obstructed. The simplest way to check this is to pull the needle out of the patient. Normally, fat tissue present in the needle is then directly and audibly forced into the syringe because of negative pressure. If this is not the case and fat tissue obstructs the needle completely, tissue in the needle can be removed by using positive pressure in the syringe. This may result in a rather explosive evacuation (firing fat tissue) and should therefore be carried out carefully. The needle is introduced into a clean container (e.g., sputum or urine) or empty syringe: tissue is then evacuated into this container or syringe, while fixing the needle firmly to the syringe to prevent the needle leaving the syringe (firing needles).

If much arterial or venous blood enters the syringe by accident, the needle should be removed out of the body. The puncture site should be pressed for at least 1 min, and the procedure can be repeated into a different direction or at a different site. Pain is infrequent, localized, and seldom a real problem necessitating the use of more lidocaine. If bruising is suspected to be present at the end of the procedure, the patient him/herself may press the puncture site for a couple of minutes before rising from the supine position. At the end of the procedure, after sealing the two syringes, both puncture sites should be inspected for the development of significant bruising. If these safety precautions are taken routinely, the chance of severe bleeding or infections is very small. In about 2,000 procedures in our hospital, we observed twice a bleeding complication necessitating surgical assistance to find and stop the bleeding locus and once a local infection of the abdominal wall necessitating surgical drainage after incision combined with antibiotic treatment.

Congo Red Stain and Grading of Amyloid in Fat Tissue

Preparing Slides for Microscopy

After extracting the plunger, fat tissue can be collected from the syringe on an empty glass slide to separate fat tissue from accidentally obtained blood. At least four visible fragments of fat tissue (not fat droplets!) should be put on each of three glass slides (preferably with a frosted edge, which can be used to write on it with a pencil). These fragments are crushed into a single layer by squeezing a second slide placed perpendicularly to the first ones (Fig. 16.7a, b). It is important to press in the middle of the glass slides to prevent breaking of glass. The resulting six smears are marked for identification, dried in the air at room temperature for 1 h, and subsequently fixed with acetone for 10 min. After drying and fixation, all slides can be stored at room temperature until shipped to a reference laboratory for staining with Congo red and further study if positive for amyloid. Fat tissue should not be frozen before slides have been made: freezing of fresh and unfixed tissue may affect the quality of the tissue.

Fig. 16.7

(a) Perpendicularly positioned glass slides. (b) Squeezing in the middle of the glass slides

Congo Red Stain, Microscopy, and Amyloid Grading

Staining with alkaline Congo red should be performed according to the classic method described by Puchtler [11]. See Table 16.4 for a short summary. Commercial kits for Congo red stain are also available and have been used successfully, in particular in the US (MM Picken, personal communication).

Table 16.4 Alkaline Congo red stain according to Puchtler [11]

The affinity of tissue for Congo red can be analyzed by the apple-green birefringence in polarized light using a good microscope. In our institution, we use the Olympus BX 50 microscope and a strong (100 W) light source. Two investigators score the slides blinded to the clinical data and in a semiquantitative grading system (Fig. 16.2): 0 (negative, no apple-green birefringence detectable), 1+ (minute, <1 % of surface area), 2+ (little, between 1 % and 10 %), 3+ (moderate, between 10 % and 60 %), 4+ (abundant, >60 %). Because some deposits may be tiny and hardly visible in daylight conditions, the slides, ideally, should be read in the dark. Kimmich et al. show nicely how this technique reliably can be introduced and successfully implemented in another center [85].

Immunochemical Quantification of Amyloid Proteins in Fat Tissue Extracts

Aim of the fat aspiration procedure is to obtain first an adequate quantity for microscopic analysis (3 × 4 lumps with total weight about 30 mg) and further at least 30 mg of fat tissue for immunochemical quantification of the amyloid proteins. After extracting the plunger, fat tissue is collected from the syringe on an empty glass slide to separate fat tissue from accidentally obtained blood and the 12 lumps of fat are used for the smears (vide supra). Before quantification, the amount of fat is weighed to get the “wet weight.” The material is then first washed 3× in a Tris buffer supplemented with calcium to remove possible remnants of blood still present. Subsequently SAP is extracted from this solution by incubation for 24 h with a Tris buffer supplemented with EDTA and the SAP concentration can be measured in this extract by ELISA [107].

The washed fat tissue is then extracted in a solution of 6 M guanidine hydrochloride and 0.1 M Tris–HCl, pH 8.0, mixed thoroughly, and shaken overnight. The suspension is centrifuged at 10,000×g for 10 min and the supernatant fat tissue extract collected. Microtiter plates are coated with the IgG fraction of the SAA-reactive mouse monoclonal capture antibody Reu.86.5 (Hycult Biotechnology, Uden, The Netherlands). The plates are washed, followed by incubation of the samples. The plates are washed again, followed by incubation with the IgG fraction of the SAA1-reactive mouse monoclonal detection antibody Reu.86.1 (Hycult Biotechnology) coupled to horseradish peroxidase. After washing, the plates are incubated with the chromogen 3′3′5′5′tetramethylbenzidin (TMB, Carl Roth, Karlsruhe, Germany) dissolved in acetate buffer until the reaction is stopped by adding H₂SO₄. The absorption at 450–575 nm is read in a Versamax microplate reader and amyloid A protein concentrations are calculated by SOFTmax^® PRO software (Molecular Devices, Sunnyvale, USA) according to a standard curve of purified SAA. The intra-assay and interassay coefficients of variation are both less than 10 % and the lower limit of detection of the amyloid A protein in fat extract is 1.6 ng/ml extraction fluid. Amyloid A protein concentration reference range of patients without AA amyloidosis is <11.6 ng/mg fat tissue [10, 61]. Recently Hycult has introduced a commercial SAA ELISA kit [108].

In a similar way, concentrations of other important amyloid proteins such as TTR and immunoglobulin light chains kappa and lambda can be measured using ELISA and nephelometry, respectively [64, 65]. Table 16.2 shows the cutoff values that were found in all four assays for the main amyloid types. However, after using the assays for a couple of years the new data have been analyzed to adapt the cutoff values for optimum prediction in daily practice (see Fig. 16.8). PPVs and NPVs have been based on 1886 routine fat tissue samples for AA, 742 samples for ATTR and 596 samples for AL-lambda and kappa immunochemical typing. Finally, 194 routine fat tissue samples have been used to test the assay. No amyloid was found in 34 samples and the remaining 160 amyloid-containing samples were analyzed for all four types. Typing was successful in 58 of 59 CR4+ samples (98 %), 39 of 45 CR3+ samples (87 %), 5 of 25 CR2+ samples (20 %) and 4 of 32 CR1+ samples (13 %). Figure 16.8 shows the decision tree currently used in Groningen for immunochemical typing of amyloid found in fat tissue.

Fig. 16.8

Cutoff values of the concentration of AA, TTR, and kappa/lambda light chain ratios in fat tissue for optimum prediction of the type of amyloid as being used in our center in Groningen (GUARD) in daily practice. PPV is positive predictive value, NPV is negative predictive value, and CR is Congo red-stained severity grading 1+ to 4+