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A reliable diagnosis of amyloidosis is usually based on a tissue biopsy. With increasing options for specific treatments of the different amyloid diseases, an exact and valid diagnosis including determination of the biochemical fibril nature is imperative. Biopsy sites as well as amyloid typing principles vary and this paper describes methods employed at some laboratories specialised in amyloidosis in Europe, Japan and USA.

Systemic amyloidosis (SA) is often diagnosed late. Combining clinical and biochemical biomarkers is necessary for raising suspicion of disease. Fine needle aspiration (FNA) of subcutaneous fat enables SA detection by Congo red staining. The luminescent conjugated probe heptameric formic thiophene acetic acid (h-FTAA) is a sensitive alternative to Congo red-staining of tissue samples. Our objective was to compare h-FTAA fluorescence with the Congo red stain for amyloid detection in FNA-obtained fat tissue. Herein, we studied samples from 57 patients with established SA (19 with AA, 20 with AL, and 18 with ATTR) and 17 age-matched controls (34-75 years). Positivity for h-FTAA was graded according to a Congo red-based grading scale ranging from 0 to 4+. Amyloid grading by both methods correlated strongly (r = 0.87). Here h-FTAA was positive in 53 of 54 Congo red-positive cases (sensitivity 98%) and h-FTAA was negative in 7 of 17 Congo red-negative controls (specificity 41%), but was also positive for 3 Congo red-negative SA cases. We conclude that h-FTAA fluorescence is more sensitive than Congo red staining in this small exploratory study of fat tissue samples, implicating potential sensitivity for prodromal amyloidosis, but is less specific for clinical amyloidosis defined by Congo red positivity. Given its simplicity h-FTAA staining may therefore be the most appropriate method for rapid screening of fat tissue samples but should presently treat grade 1+ as only suggestive, whereas 2+ or higher as positive for amyloidosis. Parallel assessment of h-FTAA and Congo red staining appears highly promising for clinical applications.

Objective: To assess the usefulness of bone scintigraphy with (99m)Technetium-hydroxymethylene diphosphonate ((99m)Tc-HDP) for the detection of cardiac involvement in a group of patients with ATTR amyloidosis in different phases of disease, to relate the findings to echocardiography, ECG and cardiac biomarkers, and to evaluate different bone scintigraphic techniques and calculation methods for quantification of the cardiac uptake and for correlation with echocardiographic features and cardiac biomarkers., Methods: Forty-one patients underwent clinical examinations, echocardiography, ECG, measurement of cardiac biomarkers and bone scintigraphy (planar imaging and SPECT-CT) and were subsequently subdivided into three groups: (1) carriers of an amyloidogenic TTR mutation, n = 11, (2) proven ATTR amyloidosis without echocardiographically-defined (mean wall thickness >12 mm) cardiac amyloidosis (AC), n = 19, and (3) ATTR amyloidosis with echocardiographically-defined cardiac amyloidosis, n = 11. Planar and SPECT-CT images were analyzed visually according to a routine scoring system (grade 0-3) and semi-quantitatively by heart-to-whole body (H/WB) and heart-to-skull (H/S) ratio on planar images and by a left ventricle-blood pool ratio on SPECT-CT images., Results: All patients with ATTR and echocardiographically-defined AC and none of the carriers showed high cardiac uptake on bone scintigraphy. Furthermore, 8 out of 19 patients with ATTR without echocardiographically-defined AC showed high cardiac uptake. Highest correlations were found between H/S ratio on planar bone scintigraphy with troponin T (r = 0.76, p < 0.0001) and H/WB ratio with left ventricular mass index (r = 0.73, p < 0.0001)., Conclusions: Bone scintigraphy with (99m)Tc-HDP may detect cardiac involvement in patients with ATTR amyloidosis prior to echocardiographic evidence of cardiac involvement. Cardiac uptake on bone scintigraphy correlates with severity of cardiac involvement using echocardiography, ECG and cardiac biomarkers. Visual grading and calculation of H/S ratio on planar imaging are the preferred methods to assess cardiac uptake.

Liver transplantation aims to halt the progression of the disease in patients with familial amyloidotic polyneuropathy (FAP) caused by hereditary transthyretin-related (ATTR) amyloidosis. Insight in health-related quality of life of these transplanted FAP-patients can be of help to optimize health care delivery. The aim of this cross-sectional study was to assess the health-related quality of life of patients with FAP long-term after transplantation. Nine patients with a post-transplant follow-up of 4 years or more were included in the study. During the annual checks, health-related quality of life was measured with the Short Form-36 (SF-36). Data were compared with non-FAP transplanted patients with the same duration of follow-up and with the normal Dutch population. Pre-transplant, all patients had signs of mild to moderate peripheral polyneuropathy. The results showed that in patients with FAP health-related quality of life was stable in the first 4 years after transplantation. The domain of physical well-being at 4 years after transplantation was significantly lower compared to non-FAP transplanted patients and control Dutch population. The domain of emotional well-being was comparable with non-FAP controls. However, on most health areas patients with FAP scored lower than the non-FAP transplanted patients and the Dutch controls. After four years, the three patients with FAP with longest follow-up (9-12 years) deteriorated in all health domains, except in self-perceived mental health. This study, including only a small number of patients with FAP, shows a relatively low health-related quality of life after liver transplantation, which may deteriorate further with longer follow-up.

To estimate the evolution of amyloid in tissue, we studied abdominal fat aspirates of cases with familial amyloidotic polyneuropathy (FAP) longitudinally at regular intervals between 1994 and 2006. In 22 cases (13 carriers and nine patients) not yet transplanted median follow-up was 3.3 years (range 0.4-11.3). We found a significant increase in the amyloid grade of fat tissue from 2+ to 4+ and from 0 to 4+ in two of three subjects with follow-ups of >7 years, after 7 and 11 years, respectively. All other subjects remained negative or did not show a significant change. In 11 liver transplant patients, follow-up with fat aspirate was available with a median duration of 3.1 years (range 1.0-10.1). A comparison was made with cardiac amyloid as judged by the cardiac septum diameter and the serum NT-ProBNP (N-terminal pro-B-type natriuretic peptide) level. No stable increase of amyloid in fat was seen in any patient. A stable decrease of amyloid grade was seen in one patient 5 years after transplantation. In contrast, the cardiac septum diameter increased >or=4 mm in six of the 11 transplant patients. Our study shows the diagnostic utility of a regularly repeated fat aspirate in carriers at risk for the development of ATTR amyloidosis. Evolution of amyloid deposition in fat tissue is very gradual. After liver transplantation, amyloid deposition in fat tissue seems to stabilize and may even decrease in the long term, whereas amyloid deposition in cardiac tissue appears to be progressive.

Objective: Amyloid A protein quantification in fat tissue is a new immunochemical method for detecting AA amyloidosis, a rare but serious disease. The objective was to assess diagnostic performance in clinical AA amyloidosis., Methods: Abdominal subcutaneous fat tissue of patients with AA amyloidosis was studied at the start of an international clinical trial with eprodisate (NC-503; 1,3-propanedisulfonate; Kiacta), an antiamyloid compound. All patients had renal findings, i.e. proteinuria (> or =1 g/day) or reduced creatinine clearance (20 - 60 ml/min). Controls were patients with other types of amyloidosis and arthritic patients without amyloidosis. Amyloid A protein was quantified by ELISA using monoclonal antihuman serum amyloid A antibodies. Congo red stained slides were scored by light microscopy in a semiquantitative way (0 to 4+)., Results: Ample fat tissue (>50 mg) was available for analysis in 154 of 183 patients with AA amyloidosis and in 354 controls. The sensitivity of amyloid A protein quantification for detection of AA amyloidosis (>11.6 ng/mg fat tissue) was 84% (95% CI: 77 - 89%) and specificity 99% (95% CI: 98 - 100%). Amyloid A protein quantification and semiquantitative Congo red scoring were concordant. Men had lower amyloid A protein values than women (p < 0.0001) and patients with familial Mediterranean fever had lower values than patients with arthritis (p < 0.001) or other inflammatory diseases (p < 0.01)., Conclusions: Amyloid A protein quantification in fat tissue is a sensitive and specific method for detection of clinical AA amyloidosis. Advantages are independence from staining quality and observer experience, direct confirmation of amyloid AA type, and potential for quantitative monitoring of tissue amyloid over time.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant disorder associated with more than 80 different transthyretin (TTR) mutations. The clinical features of FAP are broad and variable, but knowledge of the pattern and natural history of disease associated with particular mutations nevertheless offers the best guidance for management of individual patients, including the role and timming of treatment by orthotopic liver transplantation. FAP in association with TTR Gly47Glu has been described previously in an Italian kindred, and we report here its phenotype in 7 additional patients from Dutch, British, and American (Finnish) families. Characteristic clinical features included amyloid cardiomyopathy and autonomic failure but, unusually, moderate to severe renal failure was present in more than half of the cases. Only four patients were deemed to be sufficiently fit to undergo orthotopic liver transplantation, and clinical deterioration was generally rapid. These observations support early intervention with orthotopic liver transplantation in patients with FAP associated with TTR Gly47Glu.

High dose melphalan (HDM) followed by reinfusion of autologous blood stem cells (ASCT) has been applied in AL amyloidosis. Vincristine, doxorubicin, and dexamethasone (VAD) rapidly decrease light chain production in multiple myeloma. In a Phase I/II study of VAD followed by HDM and ASCT in AL amyloidosis, toxicity, feasibility, and response to this regimen were evaluated. Over a 5-year period 38 patients with AL amyloidosis were seen of which 12 out of 18 eligible patients participated in the study. VAD induced a distinct clonal response in 50% (6/12) of the patients, but without clinical improvement. In 11 patients HDM and ASCT was applied. Six months after ASCT 78% (7/9) of the surviving patients showed partial clonal response and none responded completely. Clinical condition evidently improved in 67% (6/9) of survivors, whereby clonal response, clinical response, performance score, and SAP scintigraphs were concordant. Therefore a complete clonal response is not a prerequisite for clinical improvement. With median follow-up after ASCT of 25 months, 75% of the study group patients were alive. Mortality was strongly depending on the number of organs involved Patients treated with HDM and ASCT had better survival than those not eligible (P < 0.0005).

A Dutch family with familial amyloidotic polyneuropathy associated with the transthyretin mutation Val71Ala is described. This is the third reported family with this mutation, causing at the protein level an unstable TTR monomer and at the clinical level progressive wasting, polyneuropathy, autonomic dysfunction and vitreous opacities.

Autonomic neuropathy is a well-known and prognostically important feature of systemic amyloidosis. In other conditions, autonomic function is commonly assessed by cardiovascular reflex tests, described by Ewing, but the feasibility of these tests has not been investigated in patients with systemic amyloidosis. We studied autonomic function in amyloidotic patients using cardiovascular tests and assessed their feasibility. Patients with AA, AL and ATTR amyloidosis participated. In all patients, cardiovascular reflex testing (mental arithmetic stress test and head-up tilting, besides the Ewing-tests) was performed. Of the 46 patients included, only 28 patients could perform all 4 Ewing-tests. In particular, patients with AA amyloidosis secondary to rheumatoid arthritis could not perform standing up and the isometric handgrip test. However, when the mental stress test replaced the handgrip test and head-up tilting replaced standing up, in 45 of the 46 patients, autonomic function could be assessed with cardiovascular reflex tests. Half of the patients with AA amyloidosis had signs of autonomic neuropathy--which was more than expected. We propose to replace the isometric handgrip test with the mental arithmetic stress test and standing up with head-up tilting if a patient is not able to perform these tests.