Detection of PrP^sc in Samples Presenting a Very Advanced Degree of Autolysis (BSE Liquid State) by Immunocytochemistry

Abstract

S U M M A R YAlthough detection of the abnormal isoform of prion protein (PrP sc ), the specific feature of transmissable spongiform encephalopathies (TSEs), has been previously demonstrated on formalin-fixed autolytic tissue, no samples with autolysis as severe as tested here (i.e., liquid state) have previously been tested. It is inevitable that a small but significant proportion of brains, especially in summer due to delays in postmortem examination, undergo an extremely severe autolysis that makes samples unsuitable for diagnosis by conventional techniques. In this study, 25 bovine samples were diagnosed by applying immunocytochemistry on the corresponding liquid fraction. Four additional portions of brainstem (positive and negative sheep and cattle) were subjected to one of the autolysis regimens at 56C or environmental conditions for up to 80 days and were analyzed with the same methodology. No abnormal protein could be detected in any of the control animals. PrP sc accumulation was observed by immunocytochemistry in all cases that were positive by either immunohistochemistry on the corresponding filtrates or by Prionics Western blotting, showing an excellent agreement between the methodology assessed and these routine techniques. The results of this study demonstrate immunocytochemistry as a useful tool for diagnosis in liquid-state samples, solving a most relevant problem in BSE and scrapie epidemiology.

Neuroanatomical Distribution of Disease-Associated Prion Protein in Cases of Bovine Spongiform Encephalopathy Detected by Fallen Stock Surveillance in Japan

“…However, optimal preservation is required to ensure the identification of vacuolar changes in certain areas of the brainstem devoid of artifactual vacuolations induced by a postmortem delay of tissue sampling that led to autolysis. Although autolysis conditions have been usually encountered in field cases of BSE diagnosis, autolysis and the frozen condition do not significantly influence PrP Sc detection by immunohistochemistry [4,6,7,18]. Moreover, PrP Sc immunohistochemistry is an essential tool and may contribute to our understanding of the pathogenesis of BSE [6,12].…”

Effect of Time and Temperature on PrPCWD Immunoreactivity as Evidenced by Western Blot

“…However, it has largely been concluded that autolysis does not compromise the accuracy of assays that detect the proteinase-resistant, pathogenic isoform of the prion protein (PrP res ). 2,5,7,8,12,16 It was hypothesized that over a relatively long period of time, exposure to heat may have a more substantial effect on PrP res immunodection than previously considered. The objective of the present study was to characterize PrP CWD detectability with time in brain samples subjected to heat in vitro as determined by Western blot.…”

Extended Application of Antigen Retrieval Technique in Immunohistochemistry and in SITU Hybridization

“…Monle ó n et al 72 documented an interesting IHC result of detecting the abnormal isoform of prion protein (PrP sc ) in cattle brain tissues that had been subjected to very advanced autolysis (liquid state) using the AR treatment. They took the liquid -like animal brain tissue by a swab, and made smear onto Vectabond (Vector Laboratories, Burlingame, CA) -pretreated glass slides after dilution of the liquid tissue, followed by drying at 56 ° C for 24 h, and fi xed in 10% formalin for 1 h .…”