G. Wang et al.

Microchemical Journal 180 (2022) 107620

Fig. 2. The bloodstain data on the cotton carrier were classified according to the PCA analysis score map, PC1 load map, PLSDA analysis score map and the
evaluation index results of external validation of the model. Fig. 2a: Average spectra of the bloodstains of various species on cotton carriers. Fig. 2b: The second-
derivative average spectra of the bloodstains of various species on cotton carriers. Fig. 2c: PCA analysis load diagram of spectral data of training set. Fig. 2d:
PC1 score of PCA analysis of spectral data of training set. Fig. 2e: PLS-DA analysis classification model. Fig. 2f: PLS-DA analysis classification model and model
evaluation index of test set data.

human bloodstains were incorrectly classified, which also demonstrated
the robustness and feasibility of the human and non-human bloodstains
classification models.
3.2. Identification of human and non-human bloodstains on napkin
carriers
Similarly, we analyzed the average spectrum, and it was shown that
there was no significant difference in the spectrum peak of human, cattle
and sheep bloodstains on the napkins (Fig. 3a,b), which may be because,
the rough carrier surface is not smooth, infrared spectrum is prone to
diffuse reflection on the carrier surface and form system noise when the
mixed spectrum is detected directly. This phenomenon can easily mask
the spectral information of bloodstains, and even the spectral differences
between species. We classified napkin blood samples, similar to cotton,
as human and non-human (i.e., cattle and sheep in one category). PCA
analysis results (Fig. 3c) showed that the interpretation variation of
spectral data score plots of human and non-human was about 87% on
PC1 and PC3, with human (red mark) at the bottom and non-human
(light blue mark) at the top, and human (red mark) and non-human
(light blue mark) could not be completely separate. However, there is
an intuitive tendency to distinguish between the two. Meanwhile, in the
corresponding load diagram, we noticed that two peaks played a major
role in explaining the difference (Fig. 3d), in which 1645 cm− 1 and 1537
cm− 1 represented amide I band and amide II band respectively, sug­
gesting that the difference between human and non-human bloodstains
may be mainly due to protein changes.
Next, we chose PLS-DA to establish a classification model suitable for
human and non-human bloodstains species on napkin carriers. Four LVs
were selected to construct the PLS-DA model according to the number of
LVs with the mean value of the minimum CV and Cal classification er­
rors. In Fig. 3e, the red dotted line indicates whether the sample is
human or non-human bloodstains, the red mark above the dividing line
indicates human bloodstains, and the light blue mark below the dividing
line indicates non-human bloodstains. The classification of human blood
stains was 0.872 and 0.912 (sensitivity and specificity), respectively.
After that, we loaded the test data set into the PLS-DA model to further
test the prediction ability of the PLS-DA classification model. As shown