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We have determined patient’s amyloid subtype through immunohistochemical and proteomic analyses of formalin-fixed, paraffin-embedded (FFPE) tissue samples from two affected organs per patient. Amyloid typing, via immunohistochemistry (IHC) and laser microdissection followed by the combination of liquid chromatography with mass spectrometry (LMD-LC-MS), was performed using tissue samples of the human heart, liver, kidney, tongue, and small intestine from 11 patients, and the results were compared with clinical data. LMD-LC-MS correctly typed AL amyloidosis in all 22 FFPE tissue samples despite tissue origin. In contrast, IHC was successful only in the analysis of eight FFPE tissue samples with differences between the examined organs. In the majority of LMD-LC-MS typed samples, the level of IHC staining intensity for transthyretin and serum amyloid A was the same as that for Ig κ and Ig λ antibodies, suggesting low Ig κ or Ig λ antibodies reactivity and the additional antibody clones were essential for correct typing. Both methods used in the study were found to be suitable for amyloid typing, although LMD-LC-MS yielded more promising results than IHC.

Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.

Amyloidózy patří mezi vzácná onemocnění spojená s ukládáním depozit amyloidu ve tkáních a orgánech vedoucí k jejich dysfunkci, eventuálně selhání. Rozlišují se dvě formy tohoto onemocnění -systémová, vyznačující se postižením více orgánů a tkání, a lokalizovaná (fokální). Lokalizované formy amyloidózy nejčastěji postihují močový měchýř, kůži a plíce. Plicní amyloidózy mohou mít buď lokalizovanou podobu, nebo se jedná o postižení při systémové amyloidóze, nejčastěji AL amyloidóze, jako je tomu u difuzního alveolo-septálního plicního postižení. Další dvě formy plicní amyloidózy jsou tracheobronchiální a nodulární. Všechny tři formy bývají často detekovány náhodně, a to při provedení zobrazovacích vyšetřovacích metod z jiných příčin. Jejich prognóza je oproti systémovým amyloidózám příznivější, 5leté celkové přežití činí 90,6 %. V naší kazuistice popisujeme tři pacienty, u kterých byly diagnostikovány plicní formy amyloidózy. Ve všech případech byla diagnóza stanovena po resekci postižené části plic, po které již nebyla nutná další terapie, a u pacientů nedošlo k recidivě onemocnění.Klíčová slova: amyloidóza, alveolo-septální amyloidóza, lokalizovaná amyloidóza, nodulární plicní amyloidóza, tracheobronchiální amyloidóza. Localised forms of pulmonary amyloidosisAmyloidosis is a rare disorder caused by amyloid deposits in various organs and tissues resulting in vital organ dysfunction, eventually death. There are two forms of amyloidosis -systemic, characterized by multiple organs affected, and localized (focal). Localized forms of amyloidosis usually affect urinary bladder, skin and lungs. Pulmonary amyloidosis may be localized or systemic such as diffuse alveolo-septal pulmonary amyloidosis which usually accompanies systemic AL amyloidosis. Other two forms of pulmonary amyloidosis are tracheobronchial and nodular. All three forms are usually detected by accident when patients undergo chest examination for different reasons as most cases of pulmonary amyloidosis are asymptomatic. The prognosis of localized amyloidosis is good with 5-year overall survival being 90,6 %. In our case report we present three patients diagnosed with localized pulmonary amyloidosis at our center. In all cases the diagnoses were made following the resection of affected lung segments with no further treatment needed.