systemic AA amyloidosis with cardiac involvement

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Histopathological study of multisystemic AA amyloidosis with cardiac involvement: two fatal cases in forensic practice a

b

Alessandro Bonsignore , Raffaella Gentile , Francesco De a

Stefano & Francesco Ventura

a

a

Department of Legal and Forensic Medicine , University of Genova , Via Dé Toni 12, 16132 , Genova , Italy b

Department of Clinical Pathology , San Martino Hospital , Largo R. Benzi 10, 16132 , Genova , Italy Published online: 05 Jul 2012.

To cite this article: Alessandro Bonsignore , Raffaella Gentile , Francesco De Stefano & Francesco Ventura (2012) Histopathological study of multi-systemic AA amyloidosis with cardiac involvement: two fatal cases in forensic practice, Australian Journal of Forensic Sciences, 44:4, 371-379, DOI: 10.1080/00450618.2012.691545 To link to this article: http://dx.doi.org/10.1080/00450618.2012.691545

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Australian Journal of Forensic Sciences Vol. 44, No. 4, December 2012, 371–379


Histopathological study of multi-systemic AA amyloidosis with cardiac involvement: two fatal cases in forensic practice Alessandro Bonsignorea, Raffaella Gentileb, Francesco De Stefanoa and Francesco Venturaa* a

Department of Legal and Forensic Medicine, University of Genova, Via De´ Toni 12, 16132 Genova, Italy; bDepartment of Clinical Pathology, San Martino Hospital, Largo R. Benzi 10, 16132 Genova, Italy (Received 5 September 2011; final version received 3 May 2012) In this report, two cases of death in patients diagnosed with multi-systemic AA amyloidosis at autopsy were investigated for any medical errors that may have contributed to their deaths. The patients developed nephrotic syndrome postoperatively and following trauma. The second patient had been on hemodialysis for idiopathic kidney disease prior to the trauma she sustained. Complete autopsies with immunohistochemistry and electron microscopy were used to show the extracellular deposits of amyloid in the kidneys and also in other organs, including the heart. Cardiac involvement for this type of amyloidosis has never been reported in the literature. Post-mortem analysis showed multi-systemic AA amyloidosis as the cause of nephrotic syndrome, and ultimately the cause of worsening clinical outcomes, for the two patients. A comprehensive histopathological study led to a peculiar organ-specific observation of AA amyloidosis, which requires more thorough investigations by clinicians and must be considered in the evaluation by forensic pathologists in cases of professional liability. Keywords: multi-systemic AA amyloidosis; cardiac histopathological study; professional liability; forensic approach

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involvement;

Introduction

Amyloid is a heterogeneous group of pathogenic fibrillar and abnormally folded proteins that accumulate within tissues and organs. This is due to either excess synthesis or resistance to catabolism. These proteins share the ability to aggregate into an insoluble, cross-beta-pleated sheet of tertiary conformation. Amyloid accumulates primarily as deposits in the extracellular space in a variety of tissues. Here it produces pressure atrophy of the adjacent parenchyma. Amyloidosis may be asymptomatic1, found only as an unsuspected anatomic change, or it may be life threatening2. Its effects depend on tissue distribution and degree of involvement. Of the 60 amyloid proteins known3, at least 36 have been associated with human disease4. Among these, 15 biochemically distinct forms of amyloid are the most important and only a few of them are seen commonly. Amyloidosis is subdivided into systemic (generalized) and localized (tissue-specific) forms. It is further classified on the basis of predisposing conditions. *Corresponding author. Email: [email protected] ISSN 0045-0618 print/ISSN 1834-562X online Ó 2012 Australian Academy of Forensic Sciences http://dx.doi.org/10.1080/00450618.2012.691545 http://www.tandfonline.com


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A. Bonsignore et al.

Multi-systemic amyloidosis, which is the focus of our cases, is a rare but lifethreatening complication generally associated with chronic inflammatory conditions, such as Crohn’s disease5. Diagnosis is made on the basis of histopathological studies. Usually, routine stains show only amorphous, acellular, hyaline, or eosinophilic extracellular material. However, with special stains, particularly Congo Red6, amyloid is salmon pink, and characteristic yellow-green birefringence may be seen using polarized light. Under electron microscopy, amyloid is composed predominantly (95%) of nonbranching fibrils of indeterminate length and a diameter of 7.5 to 10 nm. Using specific antibodies in immunohistochemical analysis, it is possible to identify the specific major fibril protein. In these cases the amyloid-associated (AA) was found, which is a non-immunoglobulin acute phase protein derived from a serum precursor called SAA (serum amyloid A related) synthesized in the liver. In secondary multi-systemic amyloidosis, the organ involvement typically includes kidneys, liver and spleen7. Cardiac involvement is only found accidentally and is too often misdiagnosed, but amyloid deposits in the heart can induce arrhythmias (maybe due to a ‘sympathetic storm’)8, angina (an inflammatory process related to amyloid A protein may trigger underlying coronary heart disease)9, or restrictive cardiomyopathy with progressive cardiac dysfunction. The prognosis is poor. Reactive amyloidosis may have a slightly better prognosis, depending on the ability to control the underlying condition. In the case of complex disease, multi-systemic AA amyloidosis requires a thorough investigation in order to avoid the worsening of clinical outcomes and consequently to prevent unexpected complications and/or death. Moreover, being that this kind of amyloidosis may develop in some inherited disease such as Familial Mediterranean Fever (FMF), a correct clinical or forensic diagnosis is helpful for relatives. In this report, two cases of death in patients diagnosed with multi-systemic AA amyloidosis at autopsy were investigated for any medical errors that may have contributed to their deaths. 2. Methods and results 2.1. Cases 2.1.1.

Case 1

A 47-year-old man came to the hospital and was diagnosed with a ‘partial mechanical small-bowel obstruction’. His medical and surgical history was significant for a three-year history of Crohn’s disease and perforation of the ascending colon requiring a previous total colectomy with ileorectal anastomosis. He underwent surgery for the ileus but post-operatively he developed nephrotic syndrome (hypoproteinemia and hypoalbuminemia with proteinuria) and renal failure. Secondary amyloidosis was hypothesized as a cause of the renal failure due to the frequent association with chronic inflammatory disease. However, a few hours later he presented episodes of cardiac arrhythmias, developed septic shock, suffered cardiac arrest, and died. 2.1.2.

Case 2

A 70-year-old woman arrived at the hospital after a motor vehicle crash. The patient was still conscious (Glasgow coma scale 9) and she was immediately admitted to the


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intensive care unit under the radiologic diagnoses of ‘right frontal and temporal skull fracture with subdural extraparenchymal hemorrhage’. She underwent craniotomy in order to try the evacuation of the hemorrhage. The patient subsequently developed cardiogenic shock (blood pressure 80/55 mmHg, faint pulse with 88 beats per minute, jugular venous distention) with decreased cardiac output and fell into a coma (Glasgow Coma Scale: 4/15). Of note, the patient was receiving dialysis treatment prior to this hospitalization due to chronic idiopathic kidney failure. Her condition quickly worsened to multi-organ system failure. After a few days, she died. 2.2. Autopsy findings Complete post-mortem examinations were performed in both cases 48 hours after death. 2.2.1.

Case 1

Significant findings of the external examination included recent surgical incisions and prior surgical scars on the abdomen. The internal examination revealed bilateral pleural effusions (1400 cc in the left pleural cavity, 100 cc in the right pleural cavity), pericardial effusion (100 cc), signs of acute peritonitis with fibrosis, and evidence of previous abdominal surgery with total colectomy and ileorectal anastomosis. Furthermore, there was an intraluminal abscess of the recently shortened ileus with multiple fistulas and strongly hyperemic intestinal mucosa of some remaining loops of bowel with small yellowish diffuse plaques. In addition, splenomegaly (dimensions 15 6 10 6 4 cm3, weight 375 g) and bilateral nephrosclerosis with multiple whitish infiltrations of the renal parenchyma were identified. 2.2.2. Case 2 The external examination was significant for surgical wounds from the craniotomy performed during hospitalization. The internal examination revealed increased dimensions (14 6 13 6 8 cm3) and weight of the heart (555 g), bilateral pleural effusions (500 cc), and multiple areas of consolidation in the pulmonary parenchyma. The kidneys were atrophic (left: 65 g, right: 60 g), and the renal surface was slightly irregular. After further examination, the kidneys appeared congested with diffuse intraparenchymal whitish and gelatinous areas; the cortical thickness was reduced bilaterally while the medullary substance was sclerotic and disrupted. A marked cerebral edema was evident and a herniation was under suspicion. No ischemic changes were present. 2.3.

Histological studies and findings

Formalin-fixed, paraffin embedded tissues were sectioned at 4 mm and stained with hematoxylin-eosin (H&E). 2.3.1.

Case 1

Abdominal viscero-visceral adhesions with neutrophil infiltration were found. Arterial vascular structures showed hyaline wall thickening attributable to amyloid


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deposition. The microscopic study of the other organs revealed multi-systemic amyloidosis involving heart, liver, spleen, and kidneys. Examination of the heart (Figure 1(a)) showed thickened vessels due to an amyloid-looking homogeneous, eosinophilic and amorphous material, vascular congestion, and, in some samples, patchy perimyocyte deposits with signs of cellular damage, such as atrophy and fibrosis, next to accumulations of amorphous eosinophilic substance. The liver (Figure 1(b)) presented with milder chronic inflammatory infiltrates, arterial vessels with thickening of the wall, minimal focal hepatocyte steatosis, and vascular and sinusoidal congestion. In the spleen, the parenchyma had been replaced by nodules of amyloid in the interstitium (‘lardaceous spleen’) and in the arterial vessel walls. The kidneys showed bilaterally eosinophilic glomeruli, some of these with amyloid deposits in the mesangium region and in the arterial vessel subendothelium; there were also tubular ectasia with proteinaceous material in the lumen and mild-intermediate grade of interstitial inflammatory infiltrates. The examination of the remaining histological samples was without remark. Amyloid presence was confirmed by positive Congo red staining (Figures 2(c) and 2(d)), immunohistochemical stain using antiserum against AA amyloid protein combined with immunoperoxidase (Figure 3: heart), and electron microscopy (data not shown). The cause of death was attributed to septic shock due to peritonitis in a patient affected by multi-organ failure with predominant cardiac and renal involvement secondary to systemic amyloidosis.

Figure 1. Multi-systemic amyloidosis (H&E 40X). Case 1: (a) heart and (b) liver; Case 2: (c) spleen and (d) kidneys.



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Figure 2. Multi-systemic amyloidosis (Congo Red staining 406). Case 2: (a) heart and (b) liver; Case 1: (c) spleen and (d) kidneys.

Figure 3. Infiltration of AA protein shown with immunohistochemical stain (monoclonal antihuman amyloid A component, combined with immunoperoxidase staining, Dako Diagnostics): Heart (406) (Case 1).

2.3.2.

Case 2

Cerebral edema and diffuse pneumonia were found. Special staining confirmed the suspected diagnosis of amyloidosis, a hypothesis made on the basis of the gross


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examination of the kidneys. As in the previous case, the histological examination showed multi-systemic amyloidosis, undiagnosed prior to autopsy, involving the heart, liver, spleen, and kidneys. Tissue samples from the heart showed arterial thickening with deposits of eosinophilic material. In the liver, blood vessels and periportal spaces were characterized by the presence of the same material, identified as amyloid. The spleen was markedly congested and showed thickening of its vascular regions (Figure 1(c)). The renal parenchyma was bilaterally completely replaced by deposits of amyloid in all its structures (glomeruli, interstitium, vessels, and basement membranes) (Figure 1(d)). The examination of other histological samples was unremarkable. All these findings were confirmed by the positive results to Congo Red staining (Figures 2(a) and 2(b)), immunohistochemical dye for AA amyloid (data not shown) and electronic microscopy (Figure 4: spleen).

Figure 4. Electronic microscopy: spleen. Dense network of 10 nm rigid and non-branching filaments (TEM 500006); amyloid arranged in parallel bundles forming the so called ‘saw tooth’ (TEM 120006) (Case 2).


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The mechanism of death was referred to multi-organ failure in a patient with cranial injury, bedridden and suffering from multiple systemic amyloidosis. 3. Discussion Multi-systemic AA amyloidosis (between 36% and 45% of all types of multisystemic amyloidosis)10,11 may develop during the course of infectious diseases (e.g. bronchiectasis, pleural empyema, osteomyelitis, fistulas, TB, syphilis, leprosy), inflammatory diseases (e.g. RA, spondylitis ankylopoietica, psoriasis, Crohn’s disease, SLE), autoimmune diseases (e.g. Castleman disease), benign neoplasms (e.g. liver adenoma)12, or malignant tumors (e.g. kidney and bladder cancer, Hodgkin’s lymphoma). The pathologist can only make a definitive diagnosis of secondary amyloidosis after reviewing histological evidence and/or performing electron microscopy. It is often overlooked by the clinicians due to the non-specific symptoms and the uncommon relationship between the affected organs or systems. Moreover, the correct diagnosis is often missed because clinicians tend to focus on the predominant organ system affected instead of searching for a multi-systemic disease. Still, in cases of complex diseases, amyloidosis can be an important factor for deterioration of the patient and can represent a contributory cause to death. However, if specifically looking for it, amyloid is easily detected through basic histological techniques. This because of its histochemical ‘nonspecific’ characteristics: eosinophilia, PAS positivity, metachromasia to methyl and crystal violet, and Congo red staining. Confirmation of amyloid deposits can be obtained with Congo red dye (applegreen birefringence under polarized light), immunohistochemistry (antibodies against the major protein component of the amyloid fibril) and ultra structural study (necessary in doubtful cases). In addition, the application of proteomic methodologies has recently been proposed for diagnosing amyloidosis13. From a clinical point of view, nephrotic syndrome and chronic renal failure are the most important clinical manifestations of amyloidosis. On the other hand, cardiac involvement is the most common cause of rapid and enigmatic deterioration14 and death, especially due to congestive heart failure following restrictive cardiomyopathy and conduction abnormalities. However, this is rarely seen in cases of multi-systemic AA amyloidosis15, and no cases have been previously described in the literature similar to the ones presented. Generally, an early diagnosis of cardiac amyloidosis (too often unrecognized both clinically and at autopsy due to the lack of accurate investigations), which may improve outcomes, requires heightened suspicion and a systematic clinical approach to evaluation16. For that reason, making an accurate diagnosis and recognizing the complications of secondary amyloidosis, which is rare but still the most common type of multisystemic amyloidosis in developing countries17, is essential. In the first case, the clinicians could also have been helped in finding the diagnosis by the fact that at least 1% of patients with Crohn’s disease develop amyloidosis18,19. Knowing the diagnosis, they could have delayed surgical intervention due to the contraindication of amyloidosis20,21. For this reason, the Court considered as inadequate the behavior of physicians focusing on the lack of medical treatment for the undiagnosed multi-systemic amyloidosis.

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In the second case, instead, the Court stated that, with a timely and early detection of amyloidosis, the doctors who made the diagnosis of ‘chronic idiopatic kidney failure’ should have started a therapy sooner, which could have slowed the progression of the disease and even lead to an improvement in the patient’s clinical condition. Indeed, they were confined to diagnose a kidney disease on the basis of clinical and laboratory data without carrying out further investigations such as a biopsy. 4.

Conclusion

In both cases, a complete autopsy and histopathological (histochemical, immunohistochemical and electron microscopic) approach revealed that amyloidosis had led to a rapid deterioration of the renal function and, especially because of the heart involvement, had played a contributory but decisive role in the worsening of the clinical condition of the patients, which was already significantly compromised by intestinal complications due to Crohn’s disease in the first case and cranial trauma in the second. The authors therefore want to stress the importance of complete studies in the medico-legal field. In particular, the use of a basic microscopic analysis supported, where possible, by more sophisticated investigations, such as immunohistochemistry and confocal22 or electron microscopy, remains crucial in determining certain kinds of diagnosis23, e.g. amyloidosis, and should be an essential part of the post-mortem investigation, even though not all literature in the field of forensic medicine agrees with it24. But finally, only through thorough analysis, can the forensic pathologist find the correct and definitive diagnosis in cases with an unexplained or even seemingly apparent cause of death. In that way it was possible to answer properly to questions regarding professional liability, as in the first case, and causal link, as in the second. References 1. Mitchell RN, Kumar V, Abbas AK, Fausto N. Robbins basic pathology. 8th ed. Saunders: Elsevier; 2007. 2. Bergesio F, Ciciani AM, Manganaro M, Palladini G, Santostefano M, Brugnano R, Di Palma AM, Gallo M, Rosati A, Tosi PL, Salvadori M. Immunopathology Group of the Italian Society of Nephrology. Renal involvement in systemic amyloidosis: an Italian collaborative study on survival and renal outcome. Nephrol Dial Transplant. 2008; 23(3):941–951. 3. Mok KH, Pettersson J, Orrenius S, Svanborg C. HAMLET, protein folding, and tumor cell death. Biochem Biophys Res Commun. 2007;354(1):1–7. 4. Pettersson-Kastberg J, Aits S, Gustafsson L, Mossberg A, Storm P, Trulsson M, Persson F, Mok KH, Svanborg C. Can misfolded proteins be beneficial? The HAMLET case. Ann Med. 2008;41:1–15. 5. De Beer FC, Mallya RK, Fagan EA, Lanham JG, Hughes GRV, Pepys MB. Serum amyloid-A protein concentration in inflammatory diseases and its relationship to the incidence of reactive systemic amyloidosis. Lancet. 1982;2:231–234. 6. Puchtler H, Sweat F, Levine M. On the binding of Congo red by amyloid. J Histochem Cytochem. 1962;10:355–364. 7. Lachmann HJ, Goodman HJ, Gilbertson JA, Gallimore JR, Sabin CA, Gillmore JD, Hawkins PN. Natural history and outcome in systemic AA amyloidosis. N Engl J Med. 2007;356(23):2361–2371. 8. Falk RH, Rubinow A, Cohen AS. Cardiac arrhythmias in systemic amyloidosis: correlation with echocardiographic abnormalities. J Am Coll Cardiol. 1984;3:107.



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