A New Diazo Reagent for Specific Staining of Conjugated Bilirubin in Tissue Sections

Abstract

A new technique is presented for the specific histochemical demonstration of conjugated bilirubin in tissue sections, using the diazonium salt of ethylanthranilate. The specificity of this method was proved by using test materials and cholestatic tissue sections. There was no diffusion artifact under the prescribed working conditions. The method is applicable to fresh cryostat sections and frozen sections of cold, formol-calcium-fixed tissues. The method could not be satisfactorily applied for the staining of total bilirubin. The method using 2,4-dichloraniline remains the best available technique for the histochemical demonstration of total bilirubin, while the technique with ethylanthranilate proves to be the most specific method available for the demonstration of conjugated bile pigments.

A clinical and histochemical study of cholestasis

“…A certain amount of bile pigment can be eluted from the tissue during the routine processing for histological embedding and staining. The importance of the mode of tissue preparation has already been stressed (Biava, 1964;Nolte, 1966;Desmet et al, 1968) and seems to be particularly important in cases with slight or minimal cholestasis.…”

“…This diazonium salt proved to be of value for the histochemical demonstration of conjugated bilirubin in tissue sections (Desmet, Bullens, de Groote, and Heirwegh, 1968). At the same time a method was developed (Raia, 1967) for the demonstration of total bilirubin with the diazonium salt of 2,4dichloraniline.…”

The congenic normal R/APfd and jaundiced R/APfd-j/j rat strains: a new animal model of hereditary non-haemolytic unconjugated hyperbilirubinaemia due to defective bilirubin conjugation

“…Also, the influence of unconjugated hyperbilirubinaemia on fertility (Davis & Yeary, 1979) and the mechanism of phototherapy of neonatal jaundice (McDonagh, Palma & Lightner, 1980;McDonagh et al, 1982) could be investigated by means of this model. Furthermore, it has been useful in the development of histochemical techniques for specific staining of bilirubin conjugates (Desmet et al, 1968). In recent years, the congenitally jaundiced rat has received increased attention from investigators interested in the treatment of Leyten, Vroemen, Blanckaert & Heirwegh inborn errors of metabolism.…”

Congenital Non-Hemolytic Jaundice: Crigler-Najjar Syndrome

“…The pigment was detected by using the diazonium salt of ethyl anthranilate [12]. The latter reagent has been shown not to react appreciably with large amounts o f unconjugated bilirubin in the absence of a reaction accelerator [12,28,29]. The simplest way to interpret the spectral and chromatographic observations is to postulate the excretion o f a tetrapyrrol, related to bilirubin and with a chromophoric structure very similar to that o f bilirubin.…”