G. Wang et al.
Microchemical Journal 180 (2022) 107620
Table 1
ATR-FTIR peak component assignment of bloodstains.
Wavenumber Assignment
(cm− 1)
~1084 Stretch. P = O symmetric of the > PO2− group nucleic acids,
(1100–1040) phospholipids
~1390–1380 Symmetric vibration of COO– of faffy acids and
polysaccharides
~1450 C–H bending from CH3
~1570–1510 Amide II
~1695–1620 Amide I
(Fig. 1). Therefore, we then took out the single rough carriers and
explained the results from two aspects: the classification model of
human and non-human bloodstains and the classification model of cattle
and sheep bloodstains by establishing a species identification model of
bloodstains on a single rough carriers.
3.1. Classification model of human and non-human bloodstains on cotton
carriers
We divided the bloodstain samples on the cotton cloth into two
categories, human and non-human. In Fig. 2a, average spectral analysis
showed that there were significant differences mainly in 1695–1620
cm− 1 and 1570–1510 cm− 1, namely amide I and amide II, which may
indicate that the difference between human and non-human bloodstains
on cotton carriers mainly contributed to protein. The second-derivative
average spectrum can increase the spectral resolution. In Fig. 2b, the
same result can be obtained, and there may be differences between
humans and cattle and sheep in multiple positions. PCA analysis results
showed that the interpretation variation of spectral data score plots of
human and non-human was about 97% on PC1 and PC2, and the trend
was arched, with human (red mark) at the bottom and non-human (blue
mark) at the top, and human (red mark) and non-human (blue mark)
Fig. 1. Average spectra and PCA analysis score of bloodstains data on the three carriers according to different classifications. Fig. 1a: Average spectra of the
bloodstains of various species on different carriers. Fig. 1b: Average spectra in the range of 1800–900 cm− 1. Fig. 1c: PCA score plot of all spectral data analyzed by
carrier type (red diamond: cotton; Green rectangle: glass; Blue triangle: napkin). Fig. 1d: PCA score of all spectral data analyzed by bloodstain species (light blue
marker: human; Pink star: cattle; Yellow circle: sheep).
could not be completely separated. However, there is an intuitive ten­
dency to separate the two (Fig. 2c). Meanwhile, in the load diagram
corresponding to PCA analysis (Fig. 2d), three peaks play a major role in
explaining the difference, among which 1643 cm− 1 and 1529 cm− 1
represent amide I band and amide II band respectively. It may be sug­
gested that the difference between human and non-human bloodstains
mainly lies in the change of protein. 1388 cm-1 represents the sym­
metrical vibration of COO− of fatty acid and polysaccharide, suggesting
that part of the difference between human and non-human bloodstains
may also be related to fatty acid and polysaccharide.
As an unsupervised statistical method, PCA is not as effective as PLS-
DA in classification. In order to effectively distinguish the observed
values between groups and the influencing factors that may lead to
differences between groups, we chose PLS-DA as a supervised classifi­
cation technique with partial least squares regression analysis charac­
teristics to establish a classification model suitable for human and non-
human bloodstain species in cotton carrier. 6 LVs were selected to
construct the PLS-DA model according to the number of LVs with the
mean values of the minimum CV and Cal classification errors. In Fig. 2e,
the red dotted line indicates whether the sample is human blood stain or
non-human blood stain, the red square above the dividing line indicates
human bloodstains and the blue mark below the dividing line indicates
non-human bloodstains. The classification of human bloodstains was
0.921 and 0.947 (sensitivity and specificity), respectively, indicating
that the PLS-DA classification model is a stable and reliable model.
To further test the predictive power of the PLS-DA classification
model, we loaded the test data set into the PLS-DA model. As shown in
Fig. 2f, the red dotted line is the boundary. The purple five-pointed star
above the boundary represents human bloodstains, while the green
circle below the boundary represents non-human bloodstains. The pre­
diction results showed that the classification results of the test data set
were 0.867 and 0.944 (sensitivity and specificity), respectively, which
were basically consistent with the indicators of the training data set.
Only the 2 spectrum of human bloodstains and the 3 spectrum of non-