With a few exceptions, the biochemical examination of postmortem CSF is not part of routine diagnostics in legal medicine [2]. In the clinical setting, however, the analysis of CSF is an obligatory diagnostic tool [16]. Previous have already demonstrated the equally high potential of CSF examination for postmortem issues [7, 12, 14, 17,18,19,20]. The assessment of CSF is not only important in clinical contexts but also represents an easily accessible medium with potentially diverse informative value in postmortem analysis. For example, CSF examinations can provide insights into neuropathological conditions and can also help determine the cause and time of death [2, 21,22,23]. Although postmortem CSF is attracting increasing interest, its examination sometimes requires additional and complex pre-measurement procedures. In our study, we focused on the visual appearance of CSF samples as a baseline indicator of the fluid’s potential usefulness, particularly regarding its color. We detected differences in visual appearance that correlated with PMI. Colorless CSF supernatants with lower OD values were commonly observed in cases with shorter PMI, while CSF supernatants with more intense, darker coloring and higher OD values were associated with longer PMI. The presence of pigments, such as oxyhemoglobin or bilirubin, appears to alter CSF coloration depending on PMI. This could potentially serve as a diagnostic tool in the forensic methodology spectrum for PMI determination in the future.

We used puncture of the spinal canal through the foramen magnum as a simple way of preserving our CSF samples during postmortem examination. This technique was used for all 183 samples analyzed. Alternative common procedures for CSF sampling such as lumbar puncture (LP) or aspiration of the cerebral ventricles [24] were not applied. The potential influence of the sampling technique on the visual color aspect of the CSF can therefore not be assessed.

In the study, some causes of death, such as traumatic causes, were underrepresented in the cohort. Consequently, the potential effects of trauma (accompanied by fractures) on CSF staining are more challenging to classify in comparison to cardiac deaths, which comprised the majority of cases in our study and in all subsequent autopsy cohorts. However, a significant blood admixture in the CSF was not observed in the TBI group, the single ITT case, or in previous studies conducted by our group [2]. Most of the cases investigated were male and older. No significant correlation between age or sex and the color or OD of postmortem CSF was established. In all three pediatric cases, the CSF appeared rose-colored. Intense staining of postmortem CSF was observed in both younger and older individuals. One aim of this descriptive study was to compile a representative cohort of autopsy samples with minimal exclusion criteria.

As additional information, we collected only data on sex, age, PMI, and CoD for the deceased. The influence of potential other factors (such as medication and drug abuse, time of agony, pre-existing diseases, body temperature and external factors related to the storage conditions of the bodies) on CSF coloration was not investigated. However, it is known that medication can influence the central nervous system at a cellular level [25, 26], and potentially affect the CSF as well. Certain medications can, for example, lead to increased protein levels in the CSF, which may impact its appearance in terms of color and, especially, turbidity [27]. Therefore, collecting more comprehensive data in future comparative studies would be beneficial.

Only one of the examined bodies showed early signs of putrefaction. In such cases, determining the exact PMI can be challenging and remains one of the greatest difficulties in legal medicine [28]. Consequently, a bias in the results related to PMI determination cannot be ruled out.

In the given study, postmortem CSF exhibited a highly diverse range of appearances rather than a uniform one. Our analysis identified various color spectra, which we categorized into polychromatic categories. Fine graduations in color shades were required, and the classification into these groups was somewhat subjective, although it was conducted according to the four-eyes principle. Due to practical constraints, no second or third rater was included in the study design. Alternative group allocations could be considered, and efforts should focus on maximizing reliability. Contrary to a pessimistic view that cadaveric CSF would be bloody or at least hemolytic in almost all cases, we identified 28 cases with completely colorless postmortem CSF. In these samples, the visual appearance was consistent with the physiological, crystal-clear CSF seen in living patients.

In the clinical setting, xanthochromia is often discussed in relation to the diagnosis of subarachnoid hemorrhage (SAH) based on the breakdown products of blood cells [27, 29]. In 2003, Seehusen et al. described the visual analysis of CSF and CSF supernatant, associating different color shades with various clinical diagnoses [27]. In contrast to their classification, our study identified finer gradations in the color qualities of CSF samples, leading to the grouping presented in our findings. We were unable to reproduce all of the CSF staining descriptions, such as the brownish color of CSF supernatants. It is important to note these observations were based on samples from living patients, not postmortem ones. Yellow coloring of CSF is also associated with liver failure and generalized, severe hyperbilirubinemia, and is sometimes seen in newborns due to elevated bilirubin levels [27]. Contrary to the expectation that yellow CSF would be common in liver failure, we detected this coloring in only one case of MOV with liver cirrhosis. In three other liver failure cases, the postmortem CSF had different colors. Regarding TBI, only two cases showed light yellow CSF, while cases with cerebral mass hemorrhage sometimes exhibited yellow CSF. Thus, postmortem CSF analysis was partially consistent with clinical observations. However, rose to red discolorations of postmortem CSF, which were not reported by Seehusen et al., were observed with similar frequency in cases of intracranial bleeding.

In general, we observed a wide range of CSF colors in our study. There was no correlation between CSF color or OD and CoD. Consequently, we found that nearly all color spectra could be summarized across different groups. Particularly with regard to TBI, red, blood-tinged CSF was not consistently detectable in our postmortem trauma samples. Thus, it is not possible to infer an underlying TBI solely based on reddish postmortem CSF according to our observations. Similarly, yellowish to yellow CSF did not reliably indicate intracranial bleeding or liver failure.

Our results indicated that OD increased with the intensity of CSF color. This was also true for the subjectively assessed turbidity of our CSF samples. However, we detected equally turbid but colorless CSF, which still showed low OD values. Further investigations using objective measurement methods, such as advanced photometry techniques, could validate our observations.

In summary, we focused on the visual appearance of CSF samples routinely collected during autopsy. Contrary to the pessimistic view that cadaveric CSF is predominantly bloody or at least hemolytic, many samples clear and transparent, without blood contamination. We found a highly significant correlation between CSF color / OD and the PMI. This finding is crucial, as determining the PMI remains a challenging aspect of postmortem examinations [28]. This study suggests a promising enhancement to conventional PMI estimation methods by introducing a rapid and accessible approach utilizing CSF color and OD.

Limitations

Our observations are ultimately based on the impressions gained during CSF inspection, which introduces potential subjectivity, as well as additional objective measurements of OD. It is important to note that despite careful sampling techniques, minimal blood admixture during CSF collection - and its influence on the staining of CSF supernatants – could not be definitively ruled out. Blood admixtures may have also occurred iatrogenically during autopsy, though proper centrifugation of the samples have minimized this effect. For samples in the ‘dark red’ group, we were unable to determine OD even after multiple measurements. The causes or cut-off values for CSF samples with intensive dark staining remain undetermined. Creating color tiles for each case aimed to provide visual comparability and illustrate the diversity of postmortem CSF colors. However, using a uniform grey background [15] for photographic documentation introduced a bias, a affecting the resulting color codes in by influencing the RGB component evaluation. As a result, color tiles of clear, colorless CSF samples appear greyish rather than ‘white’. This is a systematic error affecting all cases included. Further investigations could benefit from comparing CSF before and after centrifugation to clarify the influence of cellular components on the initial visible quality of CSF during and after sampling. Our study focused on illustration the immense diversity of postmortem CSF. Additional biochemical analyses were not performed and should be linked in future studies. The examination of other body fluids (e.g. vitreous humour) should be also included and will be addressed in subsequent investigations.

Key points 1.

Postmortem cerebrospinal fluid exhibits a diverse range of appearances, with varying colors, and often shows a clear appearance similar to physiological clinical samples of cerebrospinal fluid.

2.

There is a highly significant correlation between the color / optical density of the cerebrospinal fluid and the postmortem interval.

3.

No correlation has been found between the color / optical density of the cerebrospinal fluid and the causes of death.

4.

Optical density and turbidity increase with the intensity of color of the cerebrospinal fluid.