Body Fluid identifcation using spectroscopic methods.pptx

Advanced Forensic
Serology
Dr. Suchita Rawat Mphil PhD

Spectroscopic analysis of body fluids: UV
and IR
Locard's principle, "every contact leaves a trace,"
BODY FLUIDS

Reference: Zapata, F., de la Ossa,
M. Á. F., & García-Ruiz, C. (2015).
Emerging spectrometric
techniques for the forensic
analysis of body fluids. TrAC
Trends in Analytical
Chemistry, 64, 53-63.

Reference: Zapata, F., de la Ossa, M. Á. F., & García-Ruiz, C. (2015). Emerging spectrometric techniques for the
forensic analysis of body fluids. TrAC Trends in Analytical Chemistry, 64, 53-63.

• Disadvantages OF PRESUMPTIVE AND
CONFIRMATORY TESTS , such as:
• (1) most confirmatory tests (for blood and semen)
are destructive
• 2) it is necessary to apply different tests to
confirm each type of body fluid; this limitation
requires division of a sample into several
parts, and a portion of the sample having to be
kept for possible future analyses.
Trends in Analytical
Chemistry, 64, 53-63.

Spectroscopic analysis of body fluids: UV and IR

UV-Vis radiation may undergo,
absorption or fluorescence
emission.

most body fluids (e.g.,
semen, saliva, and urine)
blood
Trends in Analytical Chemistry, 64,
53-63.

Discrimination of Body Fluids using
ATR-FTIR

This Photo by Unknown Author is licensed under CC BY

Reference: Aparna, R., Iyer, R. S., Das, T., Sharma, K., Sharma, A., & Srivastava, A.
(2022). Detection, discrimination and aging of human tears stains using ATR-FTIR
spectroscopy for forensic purposes. Forensic Science International: Reports, 6,
100290.

Reference: Aparna, R., Iyer, R. S., Das, T., Sharma, K., Sharma, A., & Srivastava, A. (2022). Detection,
discrimination and aging of human tears stains using ATR-FTIR spectroscopy for forensic purposes. Forensic
Science International: Reports, 6, 100290.

Materials and method
Sample selection
Animal blood In the present study, animal blood specimens were collected from the repository of
wildlife institute of India, Dehradun. The blood samples were acquired from four species,
namely Asian Elephant, Indian Leopard, Royal Bengal Tiger, and Domestic pig. Details of
collected samples are enumerated in Table 1. 2.1.2. Human blood The Human blood samples
(n = 14) collected from superficial vein was employed to test the validation method.
Sample preparation
The blood samples were prepared by placing an aliquot of 10–20 μl of blood of each selected
species on clean and sterile glass slide and each sample was allowed to dry completely for
further analysis. Then, using sterile spatula, dried sample was scraped out and
homogeneously deposited on the surface of ATR crystal and directly analyzed for spectral
acquisition. For each selected animal blood species, three replicate spectra were acquired from
separate aliquots to check the reproducibility.

Instrumentation
For the analysis, the ZnSe (Zinc selenide ) ATR crystal face was properly
cleaned with acetone wipes. The background scan was executed without placing
any sample on the surface of ATR crystal for the spectral acquisition.
The eco- ATR FT-IR spectrometer (Bruker Alpha) enclosed with Smart Orbit, ZnSe
crystal face with OPUS (v 8.0) software was utilized for the scanning of blood
samples in the MIR spectral range that is 600–4000 cm-1. Samples were
scanned with accumulations of 24 scans at 4 cm-1 resolution.

Reference:Sharma, C. P., Sharma, S., & Singh, R. (2023). Species discrimination from blood traces using ATR FT-IR spectroscopy and
chemometrics: Application in wildlife forensics. Forensic Science International: Animals and Environments, 3, 100060
The region of amide I (1643 cm-1),
amide II (1527 cm1 ), amide III
(1229–1301 cm-1), and fibrinogen,
haptoglobin, IgA, IgG, and IgM
(1093 cm-1) are the most important
variables for the discrimination of
all selected species from blood

Reference: Sharma, C. P., Sharma, S., & Singh, R. (2023). Species discrimination from blood traces using ATR FT-IR spectroscopy and
chemometrics: Application in wildlife forensics. Forensic Science International: Animals and Environments, 3, 100060

Reference: Muro, C. K., Doty, K. C., de Souza Fernandes, L., & Lednev, I. K. (2016). Forensic body fluid identification
and differentiation by Raman spectroscopy. Forensic Chemistry, 1, 31-38.
2. Materials and methods
2.1. Sample analysis A total of 75 samples were purchased from Bioreclamation IVT, Inc.
(Westbury, NY) and Lee Biosolutions, Inc. (Maryland Heights, MO). The sample
population included peripheral blood, saliva, semen, sweat, and vaginal fluid donors (n =
15 each). Peripheral blood samples were prepared in 30 microL aliquots, while saliva,
semen, sweat, and vaginal fluid were all prepared with only 10 microL.
Samples were deposited onto individual microscope slides, which had been covered with
aluminum foil to avoid fluorescence interference [37], and allowed to dry completely.
The dried traces were approximately 15 mm2 in area. Spectra were collected using an
inVia Raman spectrometer (Renishaw, Inc., Hoffman Estates, IL) operated with WiRE 3.2
software. All samples were excited with a 785 nm wavelength laser.

Body Fluid Raman spectra
Blood  Strong peaks: at 753, 1372, 1577,
and 1620 cm-1: porphyrin and
pyrrole rings of the heme groups
appear
 Broad peak at 1245 cm-1 from
Amide III
 Sharp peak at 1002 cm-1 from
phenylalanine
Saliva
(mostly due to
amino acids)
 weak peak at 853 cm-1 from
Tyrosine
 strong peaks at 1003 and 1444 cm-
1, from phenylalanine and
tryptophan, respectively .
 Wide peak at 1655 cm-1 from
Amide I

Semen  strong peak at 715 cm-1 is due to
C–N symmetric stretching
in choline .
 strong peak at 830 cm-1 from
Fructose and tyrosine
 strong peak at 850 cm-1 from
Tyrosine, lactate and tryptophan
 peak at 958 cm-1 from citric acid
and spermine phosphate
hexahydrate
 Sharp peak at 1003 cm-1 due the
aromatic ring breathing of
phenylalanine and the C–N
stretching of urea .

Sweat  Peak due to lactate (856, 926,
1044, and 1452 cm-1)
 Peak due urea (1003 cm-1) .
Vaginal
scretions
 Peak from lactate at 853, 1126,
1450, and 1653 cm-1.
 Peak fromTryptophan at 1450 and
1339 cm-1.
 Urea peak at 541, 1003, and 1653
cm-1 .
 Phenylalanine peak at 1003 cm-1

Material and methods
2.1. Samples Human body fluids were purchased from Bioreclamation, Inc. (semen – 9
donors, East Meadow, NY) and Lee Biosolutions, Inc. (vaginal fluid – 7 donors, East St. Louis,
MO). Raman spectra of pure body fluids and their mixtures were acquired from samples dried
overnight at room temperature under ambient conditions.
Mixtures of vaginal fluid and semen were prepared using random pairs in ratios of 1.5:98.5,
3:97, 7:93, 12.5:87.5, 25:75, 50:50, 75:25, 93:7, and 97:3% and mixed thoroughly for 30 s
using a vortex.
One drop (~10 µL) was placed on a standard microscope slide covered with aluminum foil.
Aluminum foil was chosen to minimize Raman and fluorescent interference from the substrate.
2.2. Instrumentation Raman spectra were acquired using a Renishaw inVia Raman
spectrometer coupled with a research-grade Leica microscope with a 50x objective. A 785-nm
laser beam used for excitation was focused on a ~ 10-µm spot on a dry body fluid sample.
A ~ 3.5x2.5 mm sample area was scanned using Renishaw PRIOR automatic stage. Raman
spectra were recorded with a 10-second accumulation time from 80 equally spaced points on
the sample.
The instrument calibration was done using the Raman band of silicon standard at 520 cm− 1 .

Reference: Sikirzhytskaya, A., Sikirzhytski, V., Pérez-Almodóvar, L., & Lednev, I. K. (2023). Raman spectroscopy for the
identification of body fluid traces: Semen and vaginal fluid mixture. Forensic Chemistry, 32, 100468.

Biological fluid Raman spectra
Vaginal fluid 1002 cm-1 phenylalanine, urea
1045 cm-1 , 1655 cm-1 lactic acid, proteins, urea
1082, 1610 cm-1 cm-1 lactic acid
1310–1337 cm-1 Proteins
1445–1455 cm-1 lactic acid, urea
Semen 829, 983, 1616 cm-1 tyrosine
888, 958 , 1055, 1125, 1317, 1461 cm-1 Sphingomyelin (SPH)
1003 cm-1 Albumin, phenylalanine
1065 cm-1 Sphingomyelin (SPH), fatty acid
1327 cm-1Tyrosine, CH3CH2 wagging mode in purine bases of
nucleic acids
1448 cm-1 Albumin, choline
Most contributing Raman bands from the viewpoint of classification
Reference: Sikirzhytskaya, A., Sikirzhytski, V., Pérez-Almodóvar, L., & Lednev, I. K. (2023). Raman spectroscopy for the
identification of body fluid traces: Semen and vaginal fluid mixture. Forensic Chemistry, 32, 100468.

Sample preparation and Raman microspectroscopy
Blood and semen samples were purchased from several companies including
Bioreclamation, Inc., Lee Biosolutions, Inc., and Biological Specialty Corp. Blood/ semen
stains were prepared using samples from two different anonymous individuals (a Caucasian
male for semen and a Caucasian female for blood). Both donors were found to be negative for
HbsAg, HCV, HIV-1&2, syphilis and HIV-1 antigen. All samples, in volumes of 10 mL each, were
placed on microscope slides that were covered with aluminum foil to reduce fluorescence.
Mixtures were prepared with different blood/semen ratios (5:95, 10:90, 20:80, 30:70, 40:60,
50:50, 70:30, 75:25, 85:15, 85.5:12.5, 92.75:6.25, 96.875:3.125 and 98.437:1.5625) by
thoroughly shaking for 20 s. All samples were allowed to dry completely overnight.

Reference: Sikirzhytski, V., Sikirzhytskaya, A., & Lednev, I. K. (2012). Advanced statistical analysis of Raman spectroscopic
data for the identification of body fluid traces: semen and blood mixtures. Forensic science international, 222(1-3), 259-265.
Distinctive Raman bands of blood
(754, 1003, 1226, and 1619 cm-1 )
and semen (716, 830, 959, 1268,
1329, and 1671 cm-1 ) can be used
to identify their contributions in
mixtures.

Materials and methods Materials
Whole blood samples from a cow (n = 3), horse (n = 3), sheep (n = 3), pig (n = 3), rabbit (n =
3), and chicken (n = 3) were obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan).
Human blood samples (n = 5) were taken intravenously from the authors.
Whole blood samples of cat (n = 1) and dog (n = 2) were kindly provided by a veterinary clinic
(Ekinan Animal Hospital).
Whole blood samples (n = 3) of rat and mouse were kindly provided by the Department of
Experimental Animals, Interdisciplinary Center for Science Research, Organization for Research,
Shimane University.
About 100 μL of the blood was dropped onto gauze to make a bloodstain. After drying, all
Raman measurements were performed within 48 h of sample (The spectra range was 100–
2000 cm−1 using He-Cd laser light (532 nm) was used for excitation.)
For the time course change study, bloodstains from a human, rat, mouse, cow, pig, and rabbit
were left at room temperature for 3 months (1 day, 1 week, 2 weeks, 1 month, 2 months, and 3
months). The maximum sample dilution to detect Raman spectroscopy was investigated by
diluting the human blood (1:10, 1:50, 1:100, and 1:250) with saline.

McLaughlin, G., Doty, K. C., & Lednev, I. K. (2014). Discrimination of human and animal blood traces
via Raman spectroscopy. Forensic science international, 238, 91-95.
Raman peaks for blood (742, 1001, 1123,
1247, 1341, 1368, 1446, 1576, and 1619
cm−1 ) could be observed using a portable
Raman spectrometer, and human bloodstains
could be distinguished from nonhuman ones by
using a principal component analysis.

Body Fluid identifcation using spectroscopic methods.pptx

Body Fluid identifcation using spectroscopic methods.pptx

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Body Fluid identifcation using spectroscopic methods.pptx