All tests were successful and showed a positive control line, all but one (postmortem blood) blood positive secretions showed a second line for hemoglobin. Each fluid was tested as a duplicate, and the results were always identical. All samples included in this study showed a DNA concentration between 0.56 pg/µl (urine) and 7.72 ng/µl (wound crust) (Table S1). A complete DNA profile could be established for every sample containing at least 7 pg/µl DNA. With less DNA, at least a partial profile (> 1.5 pg/µl) could be generated, below this only single alleles were detected (Table S1).

Test results are displayed in the detection window and were read after 10 min. The manufacturer emphasizes that even weak lines should be considered as a positive result [10]. There are three different scenarios for a positive result (Fig. 1):

Detection window of the Seratec® PMB test with its separation section and the positions of the three red lines (C), (P) und (M)

Hemoglobin and D-dimers are positive. Three red lines can be seen, the hemoglobin line (P), the D-dimer line (M) and the control line (C).

Hemoglobin is positive. Two red lines can be seen, the control line (C) and the hemoglobin line (P).

D-dimers are positive. There are also two red lines, the control line (C) and the D-dimer line (M). Since this is an unrealistic result, it can be assumed that the sample is too highly concentrated with regard to the hemoglobin content. This phenomenon is referred to as the high dose hook effect [12].

A negative result is indicated by only one red line, the control line (C). If no line is visible, not even the control line (C), the test result is invalid (Fig. 1).

The negative controls using sterile water and the kit’s extraction buffer showed true negative results. Both menstrual blood samples demonstrated true positive results. Regarding negative reference samples, vaginal secretion, nasal secretion, saliva, urine and sperm secretion showed the expected negative results, i.e. only the control line (Table 1). EDTA blood also demonstrated a reliable result as described in scenario 2 (Table 1). In contrast, in samples containing blood (taken out of an injury), nasal blood, postmortem blood, and wound crust not only the hemoglobin line (P), but also a positive D-dimer line (M) was found in every sample and every analysis (Tables 1 and 2). This phenomenon was also described in a study by Tsai et al. (2022) [13]. Here, testing of artificially degraded blood samples and postmortem blood resulted in false positive detection of menstrual blood.

Moreover, in one of our samples (one replicate of postmortem blood), the high dose effect, i.e. the absence of the hemoglobin line (P) due to excessive hemoglobin concentration, was observed (Table 1) [12].

D-dimers are basically formed during fibrinolysis, which occurs as a counter mechanism to coagulation [9]. If coagulation is increased locally or globally due to injury, disease, or blunt trauma, fibrinolysis is increased, too. Because of this, D-dimers have been used as biomarkers in diagnostics for many years in various applications, e.g., for the detection of thrombosis. An overview is given in the review by Weitz et al. (2017) [14].

The risk that samples containing blood secured in the context of forensic genetic investigations were caused by sharp force or blunt trauma, i.e., injury-related, or originated from cadavers is very high. An increased D-dimer concentration due to previous increased blood coagulation and fibrinolysis cannot be excluded for this sample material, as demonstrated by the results of this study.

In contrast, EDTA buffered blood, as expected, showed a positive result for hemoglobin (P) and a negative result for D-dimer (M), but this preserved blood does not correspond to the typical blood in forensic genetic sample material. The EDTA in the tubes prevents coagulation of the blood and thus processes such as fibrinolysis. Accordingly, EDTA buffered blood does not reflect real conditions of the typical sample material in forensic genetics.

Regardless of the amount of used buffer (300 µl, 500 µl and 2 ml), menstrual blood samples demonstrated unambiguously results as described by scenario 1 with all three lines clearly visible after the specified diffusion time of 10 min (Table 2), which is in line with a description in the manual [10]. However, contrary to the description in the manual, blood (injury), nasal blood, and postmortem blood also displayed three lines for all buffer volumes (scenario 1, Table 2). Therefore, these samples showed false-positive results regardless of the buffer volume.

The weekly tests with dried menstrual blood provided reliable results over a period of 12 weeks. Regardless of the age of the menstrual blood, it was clearly identified by scenario 1, three red lines for hemoglobin (P), D-dimer (M) and the control line (C), as such. However, a slight decrease in the intensity of the D-dimer (M) lines with increasing time was observed (Fig. 2). It should be mentioned that one of the samples dried for 6 weeks showed an extremely weak, but still recognizable hemoglobin line (P). In general, the intensity of this band varied over time period, but was basically always positive.

Menstrual blood testing over a period of 12 weeks. The results at start time, after 6 weeks and after 12 weeks are shown as examples