Effects of Common Anticoagulants on the Visual Characteristics of Bloodstains

doi:10.12116/j.issn.1004-5619.2023.431003

Abstract

Abstract:

Objective To investigate the effects of three common anticoagulants, ethylene diamine tetraacetic acid-K₂ （EDTA-K₂）, sodium citrate and heparin sodium, on the visual characteristics of bloodstains such as patterns and colors. Methods Freshly collected blood samples were treated with EDTA-K₂, sodium citrate and heparin sodium respectively, and bloodstain samples were prepared on two objects, acrylic plate and ceramic tile. Bloodstains without anticoagulant treatment were used as the control sample. The drying time, wetting ratio, the number of cracks, the number of fragments, the central impression area ratio and the mean gray value of bloodstains were observed and recorded. Results Anticoagulants delayed the drying process of bloodstains. At 3.5 h, the wetting ratios of EDTA-K₂-, sodium citrate- and heparin sodium-anticoagulated bloodstains on the acrylic plate were 54.80%, 56.68% and 60.60%, respectively, which were higher than that of bloodstains without anticoagulant treatment （25.70%）. Anticoagulants increased the number of cracks, the number of fragments and the central impression area ratio. The changes of these three visual characteristics were most obvious in the sodium citrate-anticoagulated bloodstains, with the crack number of 0.93/mm, the fragment number of 46, and the central impression area ratio of 48.41% after drying. Anticoagulants reduced the mean gray value of bloodstains. Among them, the sodium citrate-anticoagulated bloodstains exhibited the longest time to peak in mean gray value, reading 3.5 h. Similar changes in visual characteristics were observed on the ceramic tile. Conclusion The three commonly used anticoagulants change the visual characteristics of bloodstains. It is not recommended to use anticoagulated blood as a substitute for preparing experimental samples in bloodstain pattern analysis experiments. Based on the changes in the visual characteristics during the drying process of bloodstains, the time of bloodstain deposition can be roughly estimated, and the blood scene staged with anticoagulated blood can be identified.

Key words: forensic medicine, forensic sceneology, bloodstain, anticoagulant, visual characteristics, drying time

CLC Number:

Zun-lei QIAN, Meng-qi WU, Yu SHI. Effects of Common Anticoagulants on the Visual Characteristics of Bloodstains[J]. Journal of Forensic Medicine, 2025, 41(1): 40-47，58.

Add to citation manager EndNote|Ris|BibTeX

URL: http://www.fyxzz.cn/EN/10.12116/j.issn.1004-5619.2023.431003

http://www.fyxzz.cn/EN/Y2025/V41/I1/40

Figures/Tables 10

Fig. 1 Area demarcation of bloodstains

Fig. 2 Measurement of visual characteristic parameters of bloodstains

Tab. 1 The time ranges for the bloodstains to reach each drying stage

干燥阶段	亚克力板				瓷砖
干燥阶段	无抗凝剂	EDTA-K₂	柠檬酸钠	肝素钠	无抗凝剂	EDTA-K₂	柠檬酸钠	肝素钠
内外分界出现	0.50~0.75	0.50~1.00	0.50~0.75	0.50~1.00	0.50~0.75	0.50~0.75	0.50~1.00	0.75~1.00
内外分界线固定	2.50~3.50	3.00~3.50	3.50~4.00	3.00~3.50	3.00~3.50	3.50~4.00	4.00~4.50	3.50~4.50
完全干燥	5.00~6.00	5.50~6.00	5.50~6.50	5.50~6.50	4.00~5.00	4.50~5.50	5.50~6.50	5.00~6.00

Fig. 3 The changes of the wetting ratios over time

Fig. 4 The changes of bloodstain patterns on the acrylic plate surface over time

Fig. 5 Variation of crack numbers over time

Fig. 6 The morphology of fully dried bloodstainson the acrylic plate surface

Fig. 7 Comparison of morphological features offour bloodstain residues on the acrylic plate

Tab. 2 The central impression area ratios of fourbloodstains on acrylic plates and ceramic tiles

血迹种类	亚克力板	瓷砖
无抗凝剂	7.01±1.15	7.81±1.34
EDTA-K₂抗凝	39.46±1.44^1）	35.36±0.55^1）
柠檬酸钠抗凝	48.41±3.10^1）2）	38.76±2.92^1）
肝素钠抗凝	25.30±2.21^1）2）3）	21.51±0.98^1）2）3）

Fig. 8 The changes of the mean gray values offour bloodstains over time

References 23

1	JAMES S H， KISH P E， SUTTON T P. Principles of bloodstain pattern analysis： Theory and practice[M]. Boca Raton： CRC Press，2005：1-10.
2	钱尊磊，李响，贾振军，等. 基于Image J软件的血迹遗留时间推断研究[J].中国法医学杂志，2023，38（1）：43-47. doi：10.13618/j.issn.1001-5728.2023.01.008 .
	QIAN Z L， LI X， JIA Z J， et al. Research on bloodstain age inference based on Image J software[J]. Zhongguo Fayixue Zazhi，2023，38（1）：43-47.
3	ATTINGER D， MOORE C， DONALDSON A， et al. Fluid dynamics topics in bloodstain pattern analysis： Comparative review and research opportunities[J]. Forensic Sci Int，2013，231（1/2/3）：375-396. doi：10.1016/j.forsciint.2013.04.018 .
4	HOME P H， NORMAN D G， PALMER A， et al. Quantifying forensic investigations involving bloodstain pattern analysis within the UK[J]. Forensic Sci Int，2022，339：111424. doi：10.1016/j.forsciint.2022.111424 .
5	LAAN N， COMPAIN C， SEYVE L， et al. The influence of coagulation on the drying dynamics of blood pools[J]. Forensic Sci Int，2019，305：110008. doi：10.1016/j.forsciint.2019.110008 .
6	CHUANG C K， LIN S P， LIN Y T， et al. Effects of anticoagulants in amino acid analysis： Comparisons of heparin， EDTA， and sodium citrate in vacutainer tubes for plasma preparation[J]. Clin Chem，1998，44（5）：1052-1056.
7	BERGMANN T， LEBERECHT C， LABUDDE D. Analysis of the influence of EDTA-treated reference samples on forensic bloodstain age estimation[J]. Forensic Sci Int，2021，325：110876. doi：10.1016/j.forsciint.2021.110876 .
8	KELLENI M. Anticoagulation drugs — The current state of the art[M]. London， UK： IntechOpen，2020：3-20.
9	SPARER A， SERP B， SCHWARZ L， et al. Storability of porcine blood in forensics： How far should we go?[J]. Forensic Sci Int，2020，311：110268. doi：10 .
	1016/j.forsciint.2020.110268.
10	GARCIA G C， ALVES-JÚNIOR J R F， SANTANA Á E， et al. Hematologic variables of the Arrau turtle （Podocnemis expansa） under the effects of different anticoagulants and cytologic stains[J]. Vet Clin Pathol，2021，50（2）：209-215. doi：10.1111/vcp.12960 .
11	PARSA M， HARMAND S， SEFIANE K. Mechanisms of pattern formation from dried sessile drops[J]. Adv Colloid Interface Sci，2018，254：22-47. doi：10.1016/j.cis.2018.03.007 .
12	RAMSTHALER F， KRÖLL A K， VERHOFF M， et al. Effect of anticoagulation therapy on drying times in bloodstain pattern analysis[J]. Int J Legal Med，2017，131（4）：955-961. doi：10.1007/s00414-017-1599-1 .
13	RATNOFF O D. The blood clotting mechanism and its disorders[J]. Dis Mon，1965，11（11）：1-49. doi：10.1016/s0011-5029（65）80013-x .
14	TANG C S， SHI B， LIU C， et al. Experimental characterization of shrinkage and desiccation cracking in thin clay layer[J]. Appl Clay Sci，2011，52（1/2）：69-77. doi：10.1016/j.clay.2011.01.032 .
15	CHEN R， ZHANG L， ZANG D， et al. Blood drop patterns： Formation and applications[J]. Adv Colloid Interface Sci，2016，231：1-14. doi：10.1016/j.cis.2016.01.008 .
16	PAL A， GOPE A， IANNACCHIONE G S. Hierarchical exploration of drying patterns formed in drops containing lysozyme， PBS， and liquid crystals[J]. Processes，2022，10（5）：955. doi：10.3390/pr10050955 .
17	CHOI W， SHIN J， HYUN K A， et al. Highly sensitive and accurate estimation of bloodstain age using smartphone[J]. Biosens Bioelectron，2019，130：414-419. doi：10.1016/j.bios.2018.09.017 .
18	刘小娟，杜晓琳，黄娟，等. 生物分析中溶血对检测的影响及其对策[J].中国新药与临床杂志，2017，36（4）：181-185. doi：10.14109/j.cnki.xyylc.2017.04.001 .
	LIU X J， DU X L， HUANG J， et al. Impact of hemolysis on detection in bioanalysis and its countermeasures[J]. Zhongguo Xinyao Yu Linchuang Zazhi，2017，36（4）：181-185.
19	ANNARELLI C C， FORNAZERO J， BERT J， et al. Crack patterns in drying protein solution drops[J]. Eur Phys J E，2001，5（S1）：599-603. doi：10.1007/s101890170043 .
20	BRUTIN D， SOBAC B， LOQUET B， et al. Pattern formation in drying drops of blood[J]. J Fluid Mech，2011，667：85-95. doi：10.1017/s0022112010005070 .
21	MARÍN Á G， GELDERBLOM H， LOHSE D， et al. Order-to-disorder transition in ring-shaped colloidal stains[J]. Phys Rev Lett，2011，107（8）：085502. doi：10.1103/PhysRevLett.107.085502 .
22	SOBAC B， BRUTIN D. Desiccation of a sessile drop of blood： Cracks， folds formation and delamination[J]. Colloids Surf A Physicochem Eng Asp，2014，448：34-44. doi：10.1016/j.colsurfa.2014.01.076 .
23	DUGYALA V R， LAMA H， SATAPATHY D K， et al. Role of particle shape anisotropy on crack formation in drying of colloidal suspension[J]. Sci Rep，2016，6：30708. doi：10.1038/srep30708 .

[1]	Guohua SHU. Practice and Prospect of Forensic Medicine Technology Transfer Under Policy Empowerment [J]. Journal of Forensic Medicine, 2026, 42(3): 191-195.
[2]	Xiaodan HE. Current Status Analysis and Pathway Exploration of the Technology Transfer in Forensic Medicine [J]. Journal of Forensic Medicine, 2026, 42(3): 196-206.
[3]	Qunyan LIU, Jinming YANG, Xiaoyu GUO. Strategies for Constructing a Technology Transfer System in Forensic Medicine Based on National Policies and Disciplinary Characteristics [J]. Journal of Forensic Medicine, 2026, 42(3): 207-214.
[4]	Jichao XU, Xiaohui CHEN, Qin SU, Dang’en GU, Tianchun LIN, Jianmiao ZHANG, Liang HAN, Ming CAI, Jian ZHAO, Quyi XU. Rapid Detection of Planktonic Microorganism DNA in Drowning： Technology Transfer and Application Implications [J]. Journal of Forensic Medicine, 2026, 42(3): 215-219.
[5]	Yunfeng ZHANG, Xinwei LIU, Linpei DONG, Xiaojun WU, Peng ZHAO, Jing RAN. Application of Portable Mass Spectrometry System in On-site Rapid Detection [J]. Journal of Forensic Medicine, 2026, 42(3): 224-231.
[6]	Lei TANG, Weihong XIE, Chengfei ZHUO, Chen XU, Huanhuan YAO, Xiaofei LI, Jianwei ZHANG, Yang LI, Jianbo YING. Development of a Fully Automated Blood Alcohol Analyzer and Its Application in Forensic Science [J]. Journal of Forensic Medicine, 2026, 42(3): 232-239.
[7]	Yawen XIAO, Yaoran YANG, Chongjun YANG, Yan SUN, Yue CHEN, Naihan HAN, Yuan MENG, Wenjun WANG, Qingshui REN, Jing ZHU, Tongshu ZHANG, Bing WANG, Liyan ZHANG. Application of Automatic Magnetic Solid-Phase Extraction Instrument in Toxic Substances and Illegal Drugs [J]. Journal of Forensic Medicine, 2026, 42(3): 240-246.
[8]	Mingjin YANG, Meiyun HE, Weian DU, Pengfei MEI, Xuelian CHENG, Xin LIU, Yuesen SHI, Hao JIN, Yiling HU, Meixin HE, Ling CHEN, Chao LIU. Enzyme Digestion LC-MS/MS Detection of Etomidate， Ketamine， Methamphe‑ tamine and Codeine in Hair [J]. Journal of Forensic Medicine, 2026, 42(3): 261-268.
[9]	Zheng QIAO, Hui YAN, Hang CHEN. Illegal Drug： Metonitazene [J]. Journal of Forensic Medicine, 2026, 42(3): 269-272.
[10]	Xiaotian YU. Common Problems and Recommendations for Manuscripts Submitted to Fayixue Zazhi [J]. Journal of Forensic Medicine, 2026, 42(3): 273-280.
[11]	Jianghong XIONG, Lifeng XU, Chaopeng YANG, Chengren ZHU, Dongbing PENG. Homicide Induced by Barium Chloride Poisoning： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(3): 292-294.
[12]	Shichao WU, Wei CHEN, Renjie DING, Huiyang ZHANG. Forensic Identification of a Pulverized Dismembered Case： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(3): 281-284.
[13]	Ruidi SHEN, Zhenhua QIAN. Research Progress on Mescaline and Its Analogues [J]. Journal of Forensic Medicine, 2026, 42(2): 151-158.
[14]	Yue LIU, Weiwei LIANG, Huanhui ZHU, Tianfu HE, Yuanyuan TIAN, Cong PENG, Songcai WANG. Detection of Chlormezanone in Blood Using HPLC-MS/MS Method [J]. Journal of Forensic Medicine, 2026, 42(2): 130-134.
[15]	Qingxia LIU, Di WEN, Shujin LI, Chunling MA, Bin CONG. Construction and Practice of the “Leading Goose Navigation and Four-Dimensional Synergy” Model for Cultivating Top Innovative Talents in Forensic Medicine [J]. Journal of Forensic Medicine, 2026, 42(2): 159-163.