The presented study was motivated by an autopsy case with initially unclear greenish to bluish organ discolorations. As is not uncommon in forensic autopsies, only little information on the medical history was initially provided. A subsequent retrospective case evaluation including a review of the medical records, and the literature revealed, that when it comes to green-blue discoloration of tissues or organs during autopsy, especially MB needs to be considered as a drug that could have been administered during medical therapy prior to death. Furthermore, TB and other colored drugs might cause such discolorations as well.

MB, also known as C.I. Basic Blue 9, was originally developed as a dye for the textile industry and is one of the first fully synthetic drugs to be used in medicine. Paul Ehrlich discovered the possibility of staining histological specimens with it and Robert Koch was the first to succeed in the microscopic visualization of tuberculosis bacteria with the help of MB staining [11]. Ehrlich and Guttmann first applied MB successfully as a drug for the treatment of malaria in 1891 [12]. Even though MB was later replaced by other drugs for this purpose [13], it was still used as an antimalarial drug by the US armed forces in the South Pacific until the end of World War II. However, MB was not very popular among the soldiers as it led to a blue coloration of the urine. In the memoirs of General Douglas MacArthur, commander of the Allied forces in the Southwest Pacific, it says: “Even at the loo we see, we pee, navy blue” [14]. After its use as an antimalarial drug, other medical indications followed. At the beginning of the 20th century, for example, MB was added to the medication of psychiatric patients to monitor their compliance based on the blue coloration of the urine. As an antidote, MB was initially used in the 1920s and 1930s for the treatment of cyanide poisoning [15] and later on for induced methemoglobinemia [16]. Nowadays, MB is still one of the most effective antidotes for the intravenous treatment of acquired methemoglobinemia [17, 18]. Probably the most common use of MB is as a dye in clinical examinations of the gastrointestinal and the urinary tract [19]. In surgery, MB is also used to visualize fistulous tracts, hollow organs and tissues (ureters, tumors) [20, 21].

Over the last two decades, studies in intensive care medicine have shown that MB can improve the hemodynamic situation in patients with therapy-refractory distributive shock [19, 22, 23] by reducing peripheral vasodilatation [24] and the mismatch between macro- and microcirculation [19].

Although the use of MB can improve hemodynamics in certain types of shock, no mortality benefit has yet been demonstrated in meta-analyses [25]. Surveys from the USA show that around 40% of (pediatric) intensive care physicians have already used or considered using MB in refractory shock [26]. To our best knowledge, up to now, no user data for adult intensive care medicine and no recommendations for the routine use of MB in acute shock exist in Germany. Therefore, systemic administration of MB in patients with refractory shock is presumably only used in individual cases. For the evaluated autopsy cases in which MB was administered intravenously (case 2, 3, 6, 9, 10, 11), it can be assumed that the patients received MB as ultima ratio treatment in states of shock.

During autopsies after antemortem administration of MB, green, greenish, green-blue, and turquoise discolorations of organs have been described in the literature, including the brain and the heart [2, 3], the spongiosa of the vertebrae [4], the parietal pleura [5] and the gastric mucosa [6]. Clinical observations of conspicuous color changes usually refer to a typical blue discoloration of the urine, which can also affect the stool [12]. In addition, some authors report temporary blue discoloration of the lacrimal fluid, sclera, saliva, mucous membranes and skin [14, 19, 27,28,29].

The observation at autopsy, where an initially subtle bluish discoloration of organs began to darken after a few seconds and turned into a strong green-blue discoloration is due to an oxidation process. In the presence of oxygen, the colorless leuco-MB turns into blue MB [5, 30]. In chemistry class, this reaction is known as the “blue bottle experiment” [31]. Depending on the way of application, MB can lead to discoloration of the mucosa in the gastrointestinal tract or to discoloration of entire organs. In the evaluated autopsy cases after clinically documented administration of MB, discoloration of the brain and heart were predominantly observed. In addition, the serosa of the lungs and pancreas were affected in one case each (Table 1). In conventional histological examinations, the autoptically visible staining by methylene blue is usually no longer detectable [5]. In the literature, only an implied faint visibility is described when using a special fixation technique [32]. Due to the retrospective nature of the study, the potential presence of MB in the tissues could not be proven by histological or toxicological examinations [4], which obviously is a limitation of the study. However, considering the literature and the clinical context, the authors have no reasonable doubt about the administration of MB as the cause of the discolorations. Beyond that, the study provides an indication of the visual detectability period of MB at autopsy. The reviewed cases included single and multiple administrations of MB, with the last administration having taken place at least 1 h and at most 5 h before death. A reliable visual post-mortem detectability period cannot be derived from the small number of cases, but case 11 may serve as an indication for the upper limit: The time of administration could not be narrowed down more exactly than less than 24 h before death and there was no more organ staining, but still a blue coloration of the urine.

TB, also known as tolonium chloride or C.I. Basic blue 17, is an acidophilic metachromatic blue dye that has been known for various medical applications since its discovery by William Henry Perkin in 1856. However, TB is mainly found in the dye industry [33]. In medicine, TB is often used for in-vivo and in-vitro staining and visualization of tissue structures. It selectively stains acidic tissue components and has a high affinity for nucleic acids and thus accumulates in cell nuclei of tissues with a high DNA and RNA content [34]. The fact that dysplastic and neoplastic cells can contain quantitatively more nucleic acids than cells of healthy tissue led to the use of TB for staining mucosal epithelia in vivo as early as the 1960s [33]. In Germany, TB is approved for vital staining in chromoendoscopy, chromolaparoscopy, for intraoperative vital staining of epithelial corpuscles for the visualization of fistula ducts and as an antidote for severe methemoglobinemia [35]. In the forensic context, TB can be used in gynecological examinations to detect injuries [36].

In case 1 and case 5, TB has been given PO during colonoscopy. Additionally, in case 1, TB was administered IV. There is no data available that would support systemic administration of TB in a state of shock. While medical MB was temporarily unavailable in Germany due to supply shortages, TB has been used as an alternative antidote to MB [37]. This alternative treatment might have been the reason for IV administration in case 1, where TB has presumably been used as a substitute for MB in a state of shock.

The evaluated autopsy cases and the clinical literature [33] suggest that TB, like MB, can also lead to similar discolorations of organs at autopsy. Local mucosal staining, e.g. during endoscopic procedures, is clinically intended [35] and well known. After systemic administration of TB however, to our best knowledge, organ discolorations at autopsy have not been reported yet. The reason for this may be the rare systemic use of TB, as it has a more limited clinical spectrum of therapeutic indications than MB.

In the three remaining cases without antemortem administration of MB or TB, the cause of death was intoxication with colored drugs or cleaning products: In two cases (case 7, 8) a mixed intoxication with Rohypnol® and in one case (case 4) an intoxication with a formalin-containing cleaning agent with blue warning color.

Rohypnol® tablets contain the intermediate-acting benzodiazepine flunitrazepam. In the past, Rohypnol® has been used illegally as an aid to sexual assault (known as a “date rape drug”) and was unknowingly given to victims in their drinks. Therefore, the manufacturer added the blue dye indigocarmine (C.I. Acid Blue 74, E 132) to the tablets which turns clear liquids into blue in order to warn potential victims. The ability of this dye to also stain the gastric mucosa blue has already been described in the literature [10]. In cases 7 and 8, it must be assumed that the discoloration of the gastric wall and the doudenal mucosa observed on autopsy is due to the blue dye indigocarmine.

In case 4, the blue warning color (dye: Acid Blue 9, E 133, Brilliant Blue FCF) in the detergent consumed obviously led to an isolated discoloration of the urinary bladder mucosa. The gastrointestinal tract was not or no longer affected, and no other organs showed any suspicious discoloration. As described in a case report, the ingestion of this dye, which is also used as a food additive, can lead to a green discoloration of the urine [35]. Therefore, it can be assumed that the blue dye of the detergent led to an isolated discoloration of the urinary bladder.