The present study revealed that blue Azan-stained hyaline in splenic arterioles is increasingly frequent with age, and red Azan-stained hyaline is found frequently in cases of circulatory disease and drowning and asphyxia. Many previous studies have reported that the incidence of hyaline change increases with age [3–5, 28], similar to the blue pattern shown in our study. Since those researchers used mainly periodic acid-Schiff (PAS) as a histochemical method to observe hyaline, their hyaline may have been stained blue if they had alternatively used the Azan stain. The blue color on Azan staining generally indicates that hyaline has formed gradually over prolonged periods [18–21], consistent with our observation of the blue pattern more frequently in older age groups in the present study. The association between this type of arteriolar hyaline and atherosclerotic change/consequences prevalent in elderly has been investigated previously [29–31]. Plesea et al. revealed that areas of hyaline material in the vascular wall are points of low resistance, and therefore possible site of wall rupture in the case of abrupt increase of blood pressure, resulting in hypertensive intracerebral hemorrhage .
The red color on Azan staining was unrelated to age and was found frequently among rapidly fatal cases such as the circulatory disease group and the drowning and asphyxia group. This color is principally reflected by fibrin contained within hyaline change [18–21]. The fibrin is generated due to activation of the coagulation cascade and can be formed in a short time. That is, the red pattern on Azan staining indicates the presence of fibrin in hyaline change, suggesting that the hyaline was recently generated. Therefore, the red pattern described in our study was likely generated just prior to the fatal accident, leading to its frequent appearance in the rapidly fatal cases. Unlike previous reports, this study was designed for forensic autopsy cases and contains many rapidly fatal cases. This characteristic of the study population may have led to the discovery of the new significance of the red pattern in autopsy.
Arteriolar hyaline change has been assumed to derive from plasma protein leakage across injured endothelial cells generated as a result of a general process, probably hemodynamic, e.g., hypertension, rather than as a consequence of any particular disease state [2, 6, 9, 10, 12]. This generation process is consistent with our observation of the red hyaline among all cause-of-death groups, although our results also seem to have an association with circulatory diseases. Feigin et al. investigated the significance of the hyaline colors of cerebral arteries using the tinctorial properties of the Azan stain as in our study . These authors termed red hyaline “the fibrinoid form of hyaline” and reported that the form was most specifically related to hypertension and frequently observed in cerebral hemorrhage. The red pattern in our study is equivalent to Feigin’s “fibrinoid form” and was probably produced through endothelial damage due to rapid elevation of blood pressure induced by stress immediately prior to death. The central arteries and penicillar arterioles of the spleen were the vessels examined in this study. These vessels are among the terminal arterioles distributed throughout the body. However, we could not identify the same lesion in any other organs despite detailed microscopic examination. The distinctive vascular network of the spleen [26, 27] may influence the formation of the red pattern. A further examination of the mechanism for the formation of the red pattern is beyond the scope of the present report, but will be of interest for future studies.
This study demonstrates that the red pattern of Azan staining may help pathologists determine the cause of death. Distinguishing hypothermia from acute coronary syndrome as the cause of death after cold exposure is occasionally difficult in practical situations because no established pathological findings of death from acute coronary syndrome within several hours are available. However, when splenic arteriolar hyaline shows the red pattern with Azan staining, we can infer that the cause of death is acute coronary syndrome rather than hypothermia, because the frequency of the red pattern is significantly higher in circulatory diseases compared to hypothermia (Figure 3, right column). Although the determination of the cause of death should be, of course, based on overall evidence, our results could provide a diagnostic clue in a challenging case to pathologists.
The presence of red pattern, although not very sensitive, was highly specific (85.7%) to rapidly fatal cases in this study. We examined a single splenic section from each patient, and the use of 2 or more sections could increase its sensitivity. However, examination of multiple sections is not a practical method. To increase the sensitivity, we propose that cases of hemorrhagic shock and those under 12 years of age be excluded from this assessment of the rapidity of death using the red pattern, because these cases did not show the pattern even when they presented a rapidly fatal course. When these cases were excluded from the evaluation of diagnostic accuracy of the red pattern for rapidity of death, each index increased, as shown in Table 2. The reason for these cases failing to demonstrate the pattern is not clear. However, the reduction of blood volume due to hemorrhage may interfere with a rapid elevation of blood pressure, and the mechanism of autoregulation in children may overcome hypertension .
The combination of the 2 colors shown in Figure 1A was excluded from detailed analyses in this study. The presence of this pattern did not significantly differ across age or cause-of-death groups (data not shown). This pattern is not useful in practical situations because it probably reflects a mixture of other patterns such as intermediate stages from the red to the blue pattern, in which materials from different stages coexist and the staining reactions are mixed. The limitations in our study are (1) a small number of cases from a single institution, (2) reliance on the presence of a witness or document for the determination of rapidity of death, and (3) no examination for the components of red Azan-stained hyaline. However, to our knowledge, this report is the first to estimate the applicability of splenic arteriolar hyaline for diagnosing the cause and rapidity of death.