Abdominal fat tissue aspirate in human amyloidosis

Abdominal Fat Tissue Aspirate in Human Amyloidosis: Light, Electron, a n d Immunofluorescence Microscopic Studies CLAUDINE ORFILA, DSc,* PATRICK GIRAUD, MD,t ANNE MODESTO, MD: AND JEAN-MICHEL SUC, MD w tron microscopic studies of lht tissue aspirates lmve been published. Congo red staining permits the visualization of amyloid, and the imnlunofhlorescence technique allows discrimination between the different types of amyloidosis. In this report we describe the findings of our study by imlntmofhlorescence with anti-AA protein antiserum and electron microscopy. T h e sensitivity o f these methods in biopsy proved amyloidotic patients and in patients at risk tor amyloidosis is considered.

Abdominal fat tissue aspirates from 12 [~atients with biopsyproved amyloidosis were investigated by different morphologic t e c h n i q u e s . By light microscopy, after staining of the fat tissue aspirates with Congo red and examination with a polarizing microscope, positive results were obtained in nine patients with amyloidosis, two of tbe three witb primary (AL) amyloidosis and s e v e n o f the nine with secondary (AA) amyloidosis. By indirect immunofluorescence, using AA antiserum, positive results were obtained in five of the nine cases of AA amyloidosis (aspirates from these five patients were positive on Congo red staining). By electron microscopy, amyloid fibrils were observed in five cases of amyloidosis (two of the AL and three of the AA type, all positive on Congo red staining). Althougb amyloid was demonstrated less frequently by immunofluorescence and electron microscopy, perhaps because of the small numbers of fat particles examined, it seems that, with Congo red staining, abdominal fat tissue aspiration is a simple and sensitive method for the diagnosis of amyloidosis. Immunofluorescence studies allow discrimination between the different types of amyloidosis. T h e method could be used in patients in whom other types of tissue biopsy are not recommended because of risks of bleeding or other problems. Hu.~t P*THOL 17:366--369, 1986.

MATERIALSAND METHODS Patients

Subcutaneous infi'aumbilical fat tissue aspiration was performed in 12 patients. In these patients previous tissue biopsies had proved amyloidosis on the basis of the green birefringence observed under polarized light after Congo red staining. Of these 12 patients, three had the primary form of amyloidosis (AL), six had the secondary form (AA), and three had familial Mediterranean fever (AA).

A method utilizing fine needle biopsy of subcutaneous fat tissue was developed for the diagnosis of amyloidosis. 1.2 Tiffs method uses Congo red st,tining and examinatioll in the polarizing microscope for green birefi'ingence for the diagnosis of amyloidosis secondary to rlleunmtoid arthritis. More recently, Libbey et al. :~ investigated 32 patients and fonnd positive results in secondary (AA) (66 per cent) and primary (AL) (95 per cent) am)'loidosis as well as in the lieredofamilial (AF) form (86 per cent). The specificity of this method seems to have been proved by these attthors. By extraction of fat tissue and imtnunodiffusion analyses, Westermark et al. l demonstrated AA protein in subcntaneous fat tissue. To the best of our knowledge, no studies employing simuhaneous immunofluorescence and elec-

Light Microscopy Subcutaneous biopsies were p e r f o r m e d in infraumbilical areas. Fat material was aspirated with a 22-gauge needle and a disposable sterile syringe with negative pressure. T h e aspirated fat contained small fragments of fat tissue and fat droplets. This material was placed as a smear on a glass slide and air-dried. After fixation in 10 per cent formaldehyde for 10 minutes, the slide was stained with 1 per cent alkaline Congo red for 15 minutes, counterstained with hematoxylin for 5 seconds, and dehydrated with alcohol and xylene according to a method described previously. 5 The slide was examined for green birefringence in a polarizing microscope.

Received from the l:actth6 tie Mfdecine, Toulouse, France. Accepted for publication August 30, 1985. * INSERM U 133, Facult6 de M6dccine Rangucil and l.aboratoire d'Optique Electronique du CNRS, Toulouse, France. t INSERM U 100, 116pital l'tlrpan, Toulouse, France. ~. Laboratoire d'hnmunologie, Universit6 Paul Sabatier, Toulouse, France. w Service de Ndphrologie, Universit6 Paul Sabatier, CIlU Rangueil, Toulouse, France. Address correspondence and reprint requests to Dr. Orfila: INSERM U 133, Facult6 de Mfdccine, 133 route de Narbonne, 311)62 Toulouse, France.

Electron Microscopy Fragments of fat tissne aspirate were fixed in ,1 per cent ghttaraldehyde, washed in HCL-cacodylate buffer, postfixed in 2 per cent osmiunl tetroxide, and embedded in Epon 812. Sections were cut on an nltramicrotome and stained with uranyl acetate and lead citrate. Thin sections were examined in an electron microscope. 366

ABDOMINAL FATTISSUEASPIRATEIN AMYLOIDOSIS(OrfiIa et al.] TABLE t.

Findings in Fat Tissue Aspirates from Patients with ProvedAmyloidosis Fat T i s s u e A s p i r a t e

O t h e r Biopsy Sites

Type o f Clinical F e a t u r e s

A m ) loidosis

Congo Red

AA

Electron Mitroscop)

Nephrotic synd,ome, h chains u r i n e Nephrotic syndrome lgG h myeloma Nephrotic syndrome, lgA K myeloma Nephrotic syndrome, Still's disease Famili:d M e d i t e r r a n e a n fever Fanfili:d M e d i t e r r a n e : m fever Ncphrotic syndrome, renal faihtre Nephrotic syndrome, chro,fic h m g disease Renal failure, ' familial M e d i t e r r a u e a , l fever Nephrotic syndrome, chronic arthritis Nephrotic syndrome, I'ellal ildCllOfill'dllOln,l Cardiomyol~athy, digestive system c a n c e r

AL

+

-

+

S

AL

+

-

+

L,M,

AL

-

-

-

S

AA

-

-

-

K,J,R

AA

-

-

-

S,Mu

AA

+

+

-

S

AA

+

+

-

K

AA

+

+

+

K

R

AA

+

-

-

M

R

AA

+

+

-

K

AA

+

+

+

K

AA

+

-

+

S

Patient 1 2 3

4 5 6

7 8 9

10 11 12

Positive

Negative R

R

R

ABI~REW.VlmXS: M, b o n e m a r r o w ; K, k i d n e y ; J , j c j u l m m ; R, r e c t u m ; S, skin; L, liver; Mu, muscle.

Immunofluorescence 5-*.. , \

Air-dried slides of fat tissue aspirate were incubated in a humid chamber at room temperature with A A antiserum (Atlantic Antibodies, Scarborough, Maine) for 30 minutes. After washing, slides were incubated with antigoat antiserum (Behringwerke AG, Marburg, W. Germany) conjugated with fluorescein. These sera appeared to be monospecific, yielding a single strong line against normal serum or plasma on immunoelectroplmresis. The slides were washed with phosphate-buffered saline and mounted in buffered gl).'cerol. The slides were examined in a Leitz Dialux mmroscope with an H B O 50 light source.

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RESULTS

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Light Microscopic Findings --The results are snnnnarized in Table 1. On

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Congo red staining, green birefringence was observed in connective tissue surrounding fat cells (figs. 1 and 2) in nine patients, two of the three with AL amyloidosis and seven of the ifine with A A anwloidosis. Resuhs were negative in three patients, one with AL amyloidosis and IgA K myeloma (skin biopsy proved amyloidosis), one with AA amyloidosis and Still's disease (kidney, jejunum, and rectal biopsy proved amyloidosis), and one with amyloidosis and familial Mediterranean fever (skin and muscle biopsy proved amyloidosis).

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HGtlRE t [top]. Abdominal fat tissue stained with Congo red and viewed by light microscopy. [ x 350.] FIGUI/E 2 [bottom]. Abdominal fat tissue [shown in fig, 1] stained with Congo red and viewed under polarized light. Green birefringence was seen around fat droplets. [ x 350.]

Electron Microscopic Findings Examination of fat tissue aspirates from five patients in whom specimens had stained with Congo

367

HUMAN PATHOLOGY

Volume 17, No. 4 [April 1986)

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FIGURE 3. Abdominal fat tissue showing fibrillar material in connective tissue surrounding fat droplet. [ x 24,000.]

red showed typical amyloid fibrils in connective tissue s u r r o u n d i n g fat droplets. T w o o f these patients (cases 1 and 2) lind AL amyloidosis with light chains in the urine, one patient (case 8) had nephrotic s)'nd r o m e and chronic lung disease, one patient had nephrotic syndrome and renal adenocarcinoma, and tile remaining patient had cardimnyopatl W and adenocarcinoma of tile digestive system (fig. 3).

tein was investigated in other tissue specimens fi'om these last patients. l'ositive resuhs were not observed ill any eases of AL amyloidosis. DISCUSSION

A new metltod for tile diagnosis of s)'stemic amyloidosis was proposed by Westermark and Stenkvist in 1973. e This method utilized tine needle biops)" of subcutaneous fat ill cases of secondary anwloidosis. It was fi'ee of complications and easy to perform, 6 unlike previous m e t h o d s , such as the i n t r a v e n o u s Congo red test introduced by Bennhold in 19237 and used for a long time. Biopsy specinmns from other sites, such as gingiva 8,9 or rectum, n, lmve been used, but the results can be falsely negative and the procedures can cause discomfort or risk of complications in the patients. II Renal or hepatic biopsies are more reliable but can cause bleeding in some patients. Tim examination of urinary sediment by electron microscopy was proposed for the demonstration of amyloid fibrils by Derosena et al., 12 but others have .revealed fibrils in tile urine of normal control subjects, la.vl In the first reports fine needle biopsy of subcu-

Immunofluorescence Findings Tile indirect i m m u n o f l u o r e s c e n c e technique with anti-AA antiserum was performed with all fat tissue slides. O f tile nine cases of AA amyloidosis (fig. 4), positive resuhs were observed in the five cases that had been positive on Congo red staining. The three patients with negative results were 1) a patient with A A amyloidosis and Still's disease and no positive results by light or electron microscopy but with A A protein in tile kidney (case 4); 2) a patient with amyloidosis axfd familial Mediterranean fever but no Congo red staining in abdominal fat tissue (case 5); and 3) a patient with amyloidosis and familial Mediterranean fever, with Congo red staining (case 7). The last patient (case 12) had cardiomyopathy, and specimens had stained with Congo red. No A A pro368

ABDOMINAL FAT TISSUEASPIRATEIN AMYLOIDOSIS [Orfila et al.]

FIGURE 4. Abdominal fat tissue with AA cJeposition around fat cell. (Immunofiuorescence. x 500.]

taneous fat was nsed for the detection of secondary (AA) amyloidosis in patients with rheulnatoid arthritis. ~,2.~ More recently, this method was used in the diagnosis of primary (ALl, secondary (AA), and heredofamilial (AF) amyloidosis. 3 The authors considered this method specific for the diagnosis of amyloidosis, reqtfiring no major organ or tissue biopsy. Positive resuhs were obtained in 95 per cent of patients with primary, 66 per cent with secondary, and 86 per cent with heredofamilial amyloidosis, ht our study we investigated primary and secondary forms of amyloidosis; an overall study for C o n g o red staining showed 75 per cent positivity. Compared with rectal biopsies, in two cases (cases 3 and 5) a good relation existed for negative resuhs, but in three cases (cases I, 8, and 9) rectal biopsies were negative and Congo red staining was seen in the fat aspirate. These resuhs suggested that this is a rather sensitive technique for the diagnosis of amyloidosis. Previous investigators did not use immunofhtorescence to diagnose secondary (AA) amyloidosis. Westermark et al. 'I demonstrated AA protein in subcutaneous fat by the extraction of fat tissue and double immunodiffusion analyses. In our stud)' indirect immunofluorescence with anti-AA antiserum was used in all cases; positive resuhs were obtained in five of the nine cases of AA atnyloidosis: one of the patients with negative resuhs had familial Mediterranean fever, one had digestive cancer, and in the two others positive results had not been observed on Congo red staining. No AA protein was present in the patients with AL amyloidosis. A technique described by Wright et al. 15 distinguished AA from the other amyloid components and seems to be specific for secondary (AA) amyloidosis. No previous report had demonstrated the presence of amyloid fibrils in subct~taneous fat. By elec-

tron microscop)' in our study, amyloid fibrils were shown in five cases, three of AA and two of AL amyloidosis. T h e low incidence of this demonstration could be explained by the small numbers of particles o f fat examined by this method. Moreover, Libbey et al. (1983), discussing the number of fragments of fat that must be examined for the diagnosis of amyloidosis (three to six particles can be necessary), indicated that the probability of finding amyloidosis increases with the number of samples examined. In conclusion, Congo red staining of abdominal, subcutaneous fat tissue aspirates is a simple and sensitive method for the diagnosis of amyloidosis, hnmunofluorescence studies allow discrimination between the different types of amyloidosis. Fat tissue aspiration nmy be recommended in the examination o f patients in whom other biopsies are contraindicated because of risks of complication or bleeding.

Acknowledgment. The authors are indebted to J. Quercy for technical assistance. REFERENCES 1. Westermark P: Occurrence of am)-loid deposits iq tile skill in secondar)' systemic amyloidosis. Acta Pathol Microbiol Scand [A] 80:718, 1972 2. Westermark, P, Stenkvist B: A new method for the diagnosis of systemic amyloidosis. Arch Intern Med 132:522, 1973 3. kibbey CA, Skinner M, Cohen AS: Use of abdo,niqal fat tissue aspirate in the diagnosis of systemic amyloidosis. Arch Intern Med 143:1549, 1983 4. Westermark P, Stenkvist B, NatvigJB, et al: Demonstration of protein AA in subcutaneous fat tissue obtained by fine needle biopsy. Ann Rheum Dis 38:68, 1979 5. Puchtler It, Sweat F, Levine M: On the binding of Congo red b) amyloid. J Histochem Cytochem 10:355, 1962 6. Westermark P, Stenkvist B: Diagnosis of secondary generalized amylllidosis by fine needle biopsy of the skin. Acta Med Scand 190:453, 1971 7. Bennhold tt: Uber die Ausscheidung intraven6s einverleibten kongorotes bei den ,~erschiedenen Erkrankungen, inbesondere bei Amyloidosis. Dtsch Arch Klin Med 142:32, 1923 8. Calkins E, Cohen AS: Diagnosis of amyloidosis. Bull Rheunl Dis 10:215, 1960 9. Trieger N, Cohen AS, Calkins E: Gingival biopsy as a diagnostic aid in ana)loid disease. Arch Oral Biol !: 187, 1959 10. Gafifi J, 8ohar E: Rectal biopsy for the diagnosis of amyloidosis. A m J Med Sci 240:332, 1960 11. Kyle RA, Spencer RJ, Dahlin DC: Value of rectal biopsy in the diagnosis of primary systemic amyloidosis. Am J Med Sci .- 251:501, 1966 12. Derosena R, Koss MN, Pirani CL: Demonstration in amyloid fibrils in urinary sediment. N Engl J Med 293:1131, 1975 13. Shirahama T, Skinner M, Cohen AS, et al: Uncertain value of urinary sediments in tile diagnosis of am)loidosis. N Engl J Med 297:821, 1977 14. Orfila C, de Graeve P, Guilhem A, et ah Study of light-, electron- and imlnunoflt~orescence inicroscop)' of urilmry sediment in am)loidosis. Virchows Arch [A] 379:113, 1978 15. Wright JR, Calkins E, Humphrey RL: Potassium pernmnganate reaction in am)loidosis. A histological method to assist in differentiating forms of this disease. Lab Invest 36:274, 1977

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