HISTOCHEMICAL AZO COUPLING REACTIONS OF THE PIGMENTS OF OBSTRUCTIVE ICTERUS AND OF HEMATOIDIN I. DIAZONIUM SALTS USED

The azo coupling reaction readily demonstrates bile pigment and hematoidin in routine paraffin sections of human postmortem and surgical material fixed in formol, alcohol, chloroform + methanol, etc. A number of freshly diazotized amines as well as several commercial stable diazonium salts have been used successfully both in acid (1 N acetic acid) and slightly alkaline (pH 7.5-8) media. Alkaline coupling yields colors with some diazotates which have been perfectly stable for 14-18 months. Acid azo coupling almost completely eliminates background staining, but the staining results appear to be somewhat less stable. However, moderately good retention of stain for 6 months has been observed. Safranin O yields dark red to purple bile casts and granules and hematoidin globules and crystals, methylene violet (C.I. 50205) gives deep violet and p-nitroaniline (fast red GG) gives deep red colors of good stability. Fast black K, fast black B, fast blue B, fast garnet GBC and fast red B have given satisfactory red colors, but stable diazotates must be reasonably fresh with preferably under 1 year of shelf storage. Ethylanthranilate, anthranilic acid and even aniline have yielded deep red stains of bile casts and hematoidin, but the azo colors are increasingly unstable in the order given, the last fading completely in 18 hr. More consistent results are obtained with ethylanthranilate when a modified Claus diazotization is used. None of the diazonium salts tested discriminated between bile casts and hematoidin. The periodic acid-Schiff glycol reaction colors bile casts red but fails to color hematoidin globules. It is thought that this reaction demonstrates the presence or absence of glucuronic acid, although of course it is not specific for that substance. For the study of hematoidin several of the red azo coupling reactions were successfully combined with a preceding Prussian blue reaction for hemosiderin. Combination of the argentaffin reaction with the Prussian blue reaction does not prove practicable in either sequence. Sulfation enhances the basophilia of bile casts, but to a lesser degree than alkaline azo coupling with sulfanilic acid.