MSc
Chemistry
Analytical Sciences Track
Literature Thesis
Determining the age of a
bloodstain
The quantitative and qualitative characteristics of different components
contained in bloodstains and their changes over time determined by
analytical chemistry techniques
by
Helci Deel s12360929
July 2020
12 ECs
February 3
rdtill July 16
thSupervisor: Examiner: Examiner:
i
Acknowledgments
I would like to take this opportunity to thank Carlos Martín Alberca for giving me a chance to work on this subject during my literature thesis. I further want to thank him for being not only being patient with me but also helping me improve my skills and regain confidence in myself. I could not have imagined having a better advisor and mentor for a person like me that
struggles with anxiety. I enjoyed working on the subject, and it not only broadened my horizon but also spiked my interest in forensic science.
Besides my advisor, I would like to thank Rob Haselberg and Arian van Asten for being my examiners. A special thanks to Rob as the track coordinator, for encouraging me to be more vocal about my anxiety and to work on my own pace. I hope that all of you will enjoy reading this literature study.
ii
Abstract
Bloodstains are one of the most crucial pieces of evidence found at crime scenes, which serves as important evidence for the forensic scientist. While determining the age of a
bloodstain is of great use for forensic scientists, there is no established method that is deemed reliable and well accepted. Lately, scientists have been taking a new approach when it comes to bloodstain age determination. In this new approach, compounds present in the blood, such as lipids, metabolites, peptides, proteins, amino acids, and other related compounds are studied,
as these compound classes might be useful as markers for bloodstain age. The literature thesis aims to summarize all the analytical techniques proposed up to date to determine bloodstain age and which compounds in bloodstains could be used in future research in search of a more reliable, well-established method for the determination of the age of a bloodstain.
Multiple compounds related to bloodstain identification were found. 62 % of them are proteins, and nearly all of the proteins are enzymes. Hemoglobin is the protein that could be traced back to the furthest age (up to 1 year). The second most analyzed compounds were DNA and RNA. DNA and RNA can provide age information up to 90 days. Other compounds analyzed were amino acids, hormones, red blood cells, alcohol, and metabolites.
The compounds were analyzed by multiple analytical techniques. 42% of the analytical methods used for bloodstain age determination are spectroscopic methods; 29% electrophoretic, 8% chromatographic methods; 5% hybrid methods, and 16 % other. These other methods are methods like the EpiTYPER.
For a more robust and precise method, future research is necessary. As a
recommendation, there should be investigated how amino acids behave in aged bloodstains, as well as for the lipids, and the nutrients.
iii
List of abbreviations
Abbreviations Full name Abbreviations Full name
(r) Correlation coefficients IgG Immunoglobulin G
8Sα3 8 S α3-Glycoprotein IgM Immunoglobulin M
AFM Atomic force spectroscopy Ile Isoleucine
AHF, FVIII Antihemophilic factor (factor VIII)
IαI Inter-α-trypsin inhibitor
Ala Alanine LDL Low-density Lipids
Arg Arginine Leu Leucine
Asn Asparagine LRG 3.1 S Leucine-rich α2-glycoprotein
Asp Aspartic Acid Lys Lysine
ATIII Antithrombin III Met Methionine
B Factor B Met-Hb Methemoglobin
B1 Basic protein B1 n.d. No data
B2 Basic protein B2 P Properdin
C1-INA C1 Esterase inhibitor PC Polymerase chain reaction
C1q C1q Component Phe Phenylalanine
C1r C1r Component PLS Partial least squares
C1s C1s Component PM Polymarker
C2 C2 Component Pmg Plasminogen
C3 C3 Component Pro Proline
C3PA C3 proactivator Pγ Post-γ-Globulin
C4 C4 Component QBC Quantitative buffy coat analysis
C4bp C4 Binding protein QBC Buffy Coat analysis
C5 C5 Component RBCs Red blood cells
C6 C6 Component RFLP Restriction fragment length
polymorphism
C7 C7 Component RMSPE Root Mean Squared Percentage Error
C8 C8 Component RNA Ribonucleic acid
C9 C9 Component SAP serum amyloid P protein
CE Serum cholinesterase SEM Scanning electron microscopy
Cp Ceruloplasmin Ser Serine
CRP C-Reactive protein sjTRECs Signal joint T-cell receptor
rearrangement excision circles
Cys Cysteine SRID Single Radial immunodiffusion
D Factor D STRs Short tandem repeats
Deoxy -Hb De-oxyhemoglobin TBG Thyroxine-binding globulin
DNA Deoxyribonucleic acid TC Total cholesterol
ELISA enzyme-linked immunosorbent assay
TC Transcortin
EPR Electron paramagnetic
spectroscopy
Tf Transferrin
FII Prothrombin TG Triglycerides
Fn Fibronectin Thr Threonine
FXIII Fibrin-stabilizing factor (factor XIII)
Trp Tryptophan
iv
GGG β2-Glycoprotein II Uv-vis Ultraviolet-visible spectroscopy
GLC Gas-liquid chromatography Val Valine
Gln Glutamine WBCs White blood cells
Glu Glutamic acid Znα2 Zn-α2-Glycoprotein
Gly Glycine α1AT α1-Antitrypsin
H Factor H α1B α1B-Glycoprotein
Hb Hemoglobin α1F α1-Fetoprotein
HbO2 Oxyhemoglobin Α1m 9.5 S α1-Glycoprotein
HC Hemichrome α1m α1-Microglobulin
HDL High density lipids α1S α1-Acid glycoprotein
His Histidine α1T α1T-Glycoprotein
HLA Human leukocyte antigen α1X α1-Antichymotrypsin
Hp Haptoglobin α2HS α2HS-Glycoprotein
Hpx Hemopexin α2M α2-Macroglobulin
HRG 3.8 S Histidine-rich
α2-glycoprotein
α2PAG Pregnancy-associated α2-glycoprotein
IEF Isoelectric focusing β2I β2-Glycoprotein I
Ig Immunoglobulins β2III β2-Glycoprotein III
IgA Immunoglobulin A β2m β2-Microglobulin
IgD Immunoglobulin D γBJP γ Bence Jones protein
Contents
Acknowledgments ... i
Abstract ... ii
List of abbreviations ... iii
1. Introduction ... 1
2. Blood composition ... 3
2.1. Blood components ... 3
2.1.1. Physical properties of blood ... 4
2.1.2. Red blood cells ... 5
2.1.3. The buffy coat ... 6
2.1.4. Plasma ... 7
2.2. Blood serum ... 15
3. Compounds being analyzed for bloodstain age determination ... 20
3.1. Proteins ... 20
3.2. DNA & RNA ... 23
3.2.1. DNA methylation assay ... 26
3.3. Amino acids determination ... 27
4. Analytical techniques for determining the age of bloodstains ... 29
4.1. Electrophoretic techniques ... 31 4.2. Spectroscopic techniques ... 33 4.3. Chromatographic methods ... 38 4.4. Other techniques ... 41 5. Discussion ... 42 6. Conclusions ... 44 7. References ... 45
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1. Introduction
Blood is essential to life; as it circulates through the human body and delivers substances. It is often one of the most important traces found at a crime scene1. Bloodstains are often of great value in forensic investigations because they provide information that is directly linked to the source. Blood analysis in the forensic field first started with gaining knowledge regarding the bloodstain donor 2. Fast forward to modern forensic sciences, and blood traces provide additional profiling information such as diet, lifestyle, age, and gender of the suspect. All this information is retrievable from a minimum amount of blood3. By estimating the age of
bloodstains, relevant information on the crime, or even the sequence of events can be revealed. This process does not require a person to be included in a central database4. By determining the age of bloodstains, a forensic scientist can give more additional information about what happened at the crime scene and how long ago it happened. To estimate the age, one can monitor something that changes over time, which can be different components of blood.
Bloodstain detection and identification are mostly achieved by examining with lights, under the microscope, by identifying chemical compounds in blood, doing spectrophotometry methods, and electrophoretic methods. Figure 1 shows the recent bloodstain analysis
approaches.
Figure 1. Bloodstain analysis approaches. Hb, hemoglobin; HLA, human leukocyte antigen; RFLP, restriction fragment length polymorphism; PC, polymerase chain reaction; STRs, short tandem repeats; PM, polymarker. Adapted from 5.
Many analytical methods have been established over the past decades to analyze the age of bloodstains6. These methods usually rely on visual inspection and analytical chemistry methods, like electrophoresis, spectroscopy, and chromatography. These methods date from 1970 to 2019. Most of the methods from the 70s were based on electrophoresis. Over the years, as analytical techniques evolved, spectroscopy and chromatography became more popular. Despite the array of techniques, current blood tests have their limitations. Some of the restrictions being that these methods take a long time to analyze and are destructive. Most of the methods cannot be completed in the field and are not accurate enough.
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Besides the problem of not having a reliable method, there are also other challenges when trying to estimate the age of blood. One of them is human variability. Concentrations of the different compounds in the blood may vary depending on gender, age, diet, and the influence of diseases. The environment also plays a role as factors like temperature, which often increases the rate of chemical reactions. Lately, scientists have been taking a new approach when it comes to bloodstain age determination. In this new approach, scientists look more into compounds present in the blood, such as lipids, metabolites, peptides, proteins, amino acids, and other related compounds to determine the age of blood.
In this work, literature published on the aging of bloodstains is described and discussed to answer the following research question: What are the best analytical tools to establish the aging process of a bloodstain? The main aim is to summarize the most important information published up to date that is related to the determination of the age of bloodstains. This literature review is done to provide researchers in the forensic field an overview of which method and which compounds are the best suited for bloodstain age analysis, as well as showing those possible compounds and the techniques that were not explored yet. The list with the different studies analyzed is proved in Appendix A. Based on the finding of online research, and this paper has been divided into three main parts. First, the blood composition is being explained, and compounds that can be analyzed in blood.
Then this paper will provide the compounds that have been investigated for bloodstain
analysis, with the bloodstain ages. Third, all analytical techniques used for bloodstain analysis will be reviewed, and the most promising ones further summarized. Besides, the result found are discussed to compare the applied analytical methodologies to suggest techniques and compounds analyze for studying the aging of bloodstains.
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2. Blood composition
When analyzing bloodstains in a forensic lab or at the crime scene, two main factors are studied. The first one is the physical characteristics like the size, shape, and distribution (how it is splattered on the surface found) of the bloodstain7. After establishing the physical
information of bloodstains, the chemical/biological properties of blood are analyzed. In this chapter, the main blood components are discussed to get a better understanding.
2.1. Blood components
Blood is a non-Newtonian pseudoplastic fluid. This fluid flows in the circulatory system and carries substances throughout the body. It has four main components: plasma, red blood cells, white blood cells, and platelets. The platelets, white and red blood cells are suspended in the plasma within the body. When blood is left standing or subjected to centrifugation, it will separate into three parts: plasma (55%), red blood cells (45%), and the buffy coat (<1%) containing the white blood cells and platelets. The various components in the blood are shown in Figure 2. The red part of the test tube indicates the red blood cells, which mostly consist of the protein hemoglobin (Hb). The buffy is indicated in the transparent section and the blood plasma in yellow. Blood plasma consists of about 92% of water and 8% of dissolved
substances like plasma proteins, hormones, electrolytes, nutrients, gases, and nitrogenous waste. For aging purposes, researchers have mainly been focusing on red blood cells and the dissolved substances primarily being proteins.
Figure 2. Blood composition after being centrifuged. After centrifuging blood, three-layer are formed: 55 % plasma, 45% red blood cells, and <1 % of buffy coat. This figure describes the chemical composition of the three layers in centrifuged blood.
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2.1.1. Physical properties of blood
One of the most noticeable physical properties of blood is its red color, which is caused by the Hb in the red blood cells. The amount of carbon dioxide or oxygen in the blood affects its color. Blood that has a bright red color is oxygenated (see Figure 3). This is mostly done in arteries. Venous blood has a lot of carbon dioxide, making it deoxygenated. Deoxygenated blood has a dark red color8.
Figure 3. A color grade of blood influenced by hemoglobin forms. Oxyhemoglobin is oxygen-rich and has a bright red color. The more oxygen, the brighter the red color. Deoxyhemoglobin is on the other side of the spectrum, being dark red and containing not oxygen.
The figure below (Figure 4) shows the degradation pathway of Hb. The figure shows the multiple forms of Hb. These forms depend on the oxidation state of the iron and what is bound on the sixth position. Furthermore, it is reported that the stiffness and adhesive force of RBCs in bloodstains increase after deposition, without the shape of the RBCs changing9.
5 | P a g e Figure 4. Degradation pathway of hemoglobin ex vivo and the color changes. It is reprinted with permission from Zadora and Menżyk 10.
When centrifuged, the buffy coat gets a dull yellow-white or brown color. The color depends on the concentration of neutrophils in the blood11. Plasma, by itself, has a yellow color. This color is caused by the pigment bilirubin, which is a degradation product of various enzymes that contain a heme12. There are two other common colors plasma occurs in: white and green. White plasma contains a high concentration of fats, while the green plasma contains a lot of ceruloplasmin. Ceruloplasmin is an enzyme that contains copper and plays a big role in the iron metabolism13.
2.1.2. Red blood cells
Red blood cells (RBCs), also known as erythrocytes, make up the majority of the three primary blood cells, about four to six million RBCs per µl blood. These donut-shaped cells are formed in the bone marrow and do not have a nucleus, meaning it does not contain DNA (deoxyribonucleic acid). Not carrying a nucleus contributes to the short life span of the RBCs of 120 days 14. RBCs transit oxygen to the cells and carbon dioxide away from them. Oxygen in blood is transported by Hb, which makes up 97% of the dried RBCs content. This protein is
6 | P a g e
built up of four polypeptide subunits: two alpha (α) and two beta (β) subunits15. Each subunit contains a heme molecule that includes an iron molecule that can bind to an oxygen molecule. Hb can also appear as Hb derivatives.
The conversion kinetics between different derivatives differs from inside the human compared to outside the body. Inside of a healthy living person, hemoglobin molecules are mainly present in two forms: De-oxyhemoglobin (deoxy-Hb) and oxyhemoglobin (HbO2), as shown in Figure 4. Degradation pathway of hemoglobin ex vivo and the color changes. It is reprinted with permission from Zadora and Menżyk 10. The iron in the heme subunit of Hb is in the ferrous state, which is bivalent (Fe2+). HbO2 molecules become superoxide anions and are diamagnetic 15-16. The HbO2 molecules can self-oxidize into methemoglobin (met-Hb), which is a reversible oxidation process of the heme iron into the ferric state (Fe3+) 17. The met-Hb cannot carry oxygen because it cannot bind to the Fe3+ 18.
Hb in bloodstains (outside of the body) saturates entirely with the oxygen of the environment to HbO2. The production of met-Hb is not reversible out of the body19. This process is irreversible due to the decreasing availability of cytochrome b5, which generally removes 95-99% of the met-Hb being produced20. The met-Hb denatures to hemichrome (HC), which is formed due to an internal conformation change to the heme group21.
2.1.3. The buffy coat
They are composed of less than 1% of the total blood volume; the buffy consists of WBCs and platelets. Quantitative buffy coat analysis (QBC), which is based on the centrifuged components, is often used to detect the blood parameters. The buffy coat can be used to reduce or concentrate large sample volumes of cells22.
2.1.3.1. White blood cells
White blood cells (WBCs) or leukocytes are formed in the lymph nodes. Unlike RBCs, they contain a nucleus meaning they contain DNA and RNA (ribonucleic acid). WBCs are responsible for keeping the organism from pathogens, the immune system. The WBCs are separated into two major categories, as mentioned in Figure 2: granular leukocytes and
agranular leukocytes. The seven different WBCs functions, developing time, and life span, are indicated in Table 1. It shows that neutrophils have the highest concentration in blood (3000-7000 µl), with the lymphocytes being the second-highest in the blood (1500-3000 µl) and has the longest lifespan (hours to years).
There are no results reported on the degradation of WBCs cells in regard to
determining the age of bloodstains. WBCs are used to analyze leukemia, which produces an abnormally large number of WBCs rapidly. It is also used to study the immune system. Bloodstain analysis often involves analyzing the WBC count. It is possible that the WBCs are not used for age determination since WBCs regulate the immune system. The WBCs also have a lower life span compared to RBCs making it more favorable to study the age of blood with RBCs instead of WBCs.
7 | P a g e Table 1. Summary of formed white blood cells. Here their function, range in blood, duration of development, and their life spam is mention.
Cell type Function Range (per µl) Duration of development Life spam Source Granular leukocytes
Neutrophils Destroys bacteria 3000 -
7000
7 to 11 days 6 hours to a few days
[8, 23]
Eosinophils Kills parasites, blocks allergic responses
100 - 400 7 to 11 days ~ 5 days [8, 23]
Basophils Releases mediators of inflammation like histamine
20 - 50 3 to 7 days Hours to few days
[8, 23]
Agranular leukocytes
Lymphocytes Forms memory cells and are essential to cytotoxic immunity
1500 - 3000
Days to week Hours to years
[8, 23]
Monocytes Develops into macrophages in tissue
100 - 700 2 to 3 days Months [8, 23]
2.1.3.2. Platelets
Just like the WBCs, platelets only make out a small fraction of blood, less than 1%. The platelets develop in five days and have a life span of five to ten days23. Platelets, also known as thrombocytes, are blood cell fragments produced by megakaryocytes. These fractions are essential mediators that trigger the mechanical pathway of the coagulation (clotting process) cascade process, which is crucial in protecting the body from excessing blood loss. Platelets stimulate primary hemostasis via three essential processes: activation, adhesion, and
aggregation14, 24. There are no results reported for measuring platelets for age determination of bloodstains, because the coagulation process happens extremely fast in bloodstains, making it not applicable for age estimation.
2.1.4. Plasma
Plasma makes out 55% of the centrifuged blood. Most of this is water (90%), while 10% consisting of solutes as plasma proteins, salts, gases, nutrients, hormones, amino acids, lipids, and more (see Figure 2). The plasma proteins degrade after deposition; thus, this can be used to study the age of bloodstains. Lately, scientists have been focusing on analyzing the other compounds in the plasma and blood in general. These compounds may chemically change in time due to the presence of different compounds, like how Hb oxidizes into HbO2. This process is due to the oxygen of the environment. The various ratios of proteins can also be used as an age indicator of bloodstains. Hormones or biomarkers, like melatonin, can be used to determine the deposition time of bloodstains relatively in 24 hours25.
2.1.4.1. Plasma proteins
Plasma protein, also referred to as blood proteins, are the proteins present in the blood plasma. These proteins are primarily synthesized in the liver and play a limited role in extracellular buffering. The plasma proteins are responsible for 20% of the nonbicarbonate buffering capacity of whole blood, and hemoglobin does the other 80%. Plasma has three major plasma protein groups albumin, globulins, and fibrinogen. Figure 5 shows the composition of plasma proteins. About 55% of the proteins are albumin. This protein is synthesized in the liver and
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serves as a binding protein. It transfers fatty acids and steroid hormones. Albumin is one of the significant proteins that can help the osmotic pressure of blood26-27. Globulins are the second most common plasma protein and has three main subgroups: alpha, beta, and gamma globulin. The alpha and beta globulin helps to transport vitamins, iron, and lipids to the cells. The gamma globulins, also known are antibodies or immunoglobulin, are involved with the immunity. The third biggest protein is fibrinogen proteins, which are responsible for blood clotting27. The other proteins present in blood plasma make up <1% of the plasma proteins. Table 2 contains all the proteins found in blood plasma, with their molecular weights and amount present in the blood. It also contains all the sub-groups of all the proteins.
Figure 5. Composition of the plasma proteins. Albumin is the most abundant class of proteins, followed by globulin and fibrinogen 55% 12% 12% 16% 5%
9 | P a g e Table 2. List of plasma proteins present in blood plasma and their subgroups. It contains the molecular weight of the proteins and the amount present in plasma. It was adapted from Putnam 28.
Protein Abbreviation Amount in plasma (mg/100ml) Molecular weight (Da) Albumin Alb 3500-5000 66,500 α-Globulins α1-Acid glycoprotein α1S 55-140 40,000 α1-Antitrypsin α1AT 200-400 54,000 α1-Fetoprotein α1F ~1µg 66,300 9.5 S α1-Glycoprotein (serum amyloid P protein) α1M, SAP 3-8 ~250,000 Globulin Gc 20-55 52,000 Ceruloplasmin Cp 15-60 132,000 3.8 S Histidine-rich α2-glycoprotein HRG 5-15 58,500 α2-Macroglobulin α2M 150-420 725,000 4 S α2, β1-Glycoprotein – Trace 60,000 α1B-Glycoprotein α1B 15-30 68,000 α1T-Glycoprotein α1T 5-12 85,000 α1-Antichymotrypsin α1X 30-60 68,000 α1-Microglobulin α1m 4-9 26,000 Zn-α2-Glycoprotein Znα2 2-15 41,000 α2HS-Glycoprotein α2HS 40-85 49,000 Pregnancy-associated α2 -glycoprotein α2PAG Trace 360,000 3.1 S Leucine-rich α2-glycoprotein LRG 2-3 49,600 8 S α3-Glycoprotein 8Sα3 3-5 220,000 Serum cholinesterase CE 0.5-1.5 348,000 Thyroxine-binding globulin TBG 1-2 54,000
Inter-α-trypsin inhibitor IαI 20-70 ~160,000
Transcortin TC ~7 55,700 Haptoglobin Hp Type 1-1 – 100-220 86,000 Type 2-1 – 160-300 ~200,000 Type 2-2 – 120-260 ~400,000 β-Globulins Hemopexin Hpx 50-115 60,000 Transferrin Tf 200-320 79,500 β2-Microglobulin β2m Trace 11,730 β2-Glycoprotein I β2I 15-30 ~48,000 β2-Glycoprotein II GGG 12-30 63,000 (C3 proactivator) C3PA – –
10 | P a g e C-Reactive protein CRP <1 105,000 Fibronectin Fn 25-40 440,000 Low-molecular-weight proteins Lysozyme 0.5-1.5 14,000 Basic protein B1 B1 – 11,000 Basic protein B2 B2 <1 8,800 0.6 S γ2-Globulin – <1 5,100 2 S γ2-Globulin – 0.1 14,000 Post-γ-Globulin Pγ – 13,260 Complement components C1q Component C1q 10-25 400,000 C1r Component C1r – 166,000 C1s Component C1s 1-2 83,000 C2 Component C2 2-3 102,000 C3 Component C3 55-120 185,000 C4 Component C4 20-50 200,000 C5 Component C5 4-15 185,000 C6 Component C6 7 105,000 C7 Component C7 6 92,500 C8 Component C8 8 163,000 C9 Component C9 23 71,000
Other complement factors
C1 Esterase inhibitor C1-INA 15-35 104,000
Factor B B – 90,000 Factor D D – 24,000 Factor H H – 155,000 C4 Binding protein C4bp – 540,000 Properdin P 2-3 220,000 Immunoglobulins Ig Immunoglobulin G IgG 800-1800 150,000 Immunoglobulin A IgA 90-450 (160,000) n Immunoglobulin M IgM 60-250 950,000 Immunoglobulin D IgD <15 175,000 Immunoglobulin E IgE <0.06 190,000
κ Bence Jones protein κBJP Trace 23,000
γ Bence Jones protein γBJP Trace 23,000
Coagulation proteins
Antithrombin III ATIII (20-40) 58,000
Prothrombin FII (5-10) 72,000
Antihemophilic factor (factor VIII) AHF, FVIII (1-2) (100,000) n
Plasminogen Pmg (6-25) 92,000
Fibrin-stabilizing factor (factor XIII)
FXIII (1-4) 320,000
Fibrinogen F1 200-450 340,000
Plasma protein profiles may be useful for bloodstain age determination, as they stay sufficiently stable over time 29. The degradation of these proteins can be studied as a mark for the age of the bloodstain. For a lot of the proteins listed in table 2, there are not many papers that link the age of a bloodstain to these papers. However, there are a lot of papers studying the stability of proteins. In 2017 Björkesten, et al. 30 studied the stability of proteins in dried blood spots. Lately, scientists in the medical field analyze dried bloodspots because it is easy, quick, and reduces cost and storage while still maintaining all the compounds in blood. Björkesten, et al. 29 studied both the direct effects on immunodetection; this is based on the drying process and the long-term storage effect. For this, they analyzed 92 proteins and
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compared the results of the proteins of the dried blood spots with the ones in blood plasma, whole blood, and diluted series of the 92 proteins. The samples we stored for up to 30 years at a temperature of 4 °C and -24 °C. These were compared with the proteins measured at -70 °C. The results indicated that the dried samples had a slight influence on the detection of the blood proteins, compared to the blood plasma and whole blood. This is in a reproducible matter. They also stated that de detection of the proteins was not significantly affected by the storage for over 30 years. The proteins that did decrease slowly during the storage time did it in a range of 10 to 50 years. There difference found in the stability of the proteins stored at the two different temperatures. The proteins stored at -24 °C were less affected than the ones of 4°C.
2.1.4.2. Gases
Plasma contains dissolved gases, with oxygen being the most abundant one. The oxygen in blood plasma is mostly bonded to the hemoglobin in RBCs. Some of the gases are directly dissolved in the blood plasma31. Carbon dioxide and nitrogen are additional blood plasma gases. While oxygen and carbon dioxide are being analyzed as part of hemoglobin and other proteins, exploring the possibilities of analyzing them as dissolved gases can be explored. So far, no studies are found regarding analyzing the dissolved gases in blood plasma.
2.1.4.3. Nutrients
The nutrients essential for the human body are classified into six classes: water, proteins (see 2.1.4.1), carbohydrates, fats (see 2.1.4.4), vitamins, and minerals. Nutrients are necessary for life and health as they help to regulate chemical processes in the body and are essential for the growth and repair of the human body. Diet and lifestyle play a significant role in the
concentrations and effects of the nutrients on the human body. While proteins can be used as a direct like to bloodstain age, other nutrients like water, vitamins, and minerals have not been researched yet. One important reason for this could that diet has to do a lot with the
concentration in people's blood, making it hard to study the age of the bloodstain. Table 3 consists of the nutrients classes found in the literature. These values are based on the minimum intake required for a healthy male and female.
Table 3. Nutrients in blood functions and daily intake concentration for males and females.
Nutrient Intake of male Intake of female Function Source
Vitamins
Vitamin A 900 µg/day 700 µg/day Plays a role in bone growth and the immune system
[32] Vitamin B1 1.2 mg/day 1.1 mg/day Helps convert food into energy.
Helps maintain the hair and skin healthy and critical for nerve functioning.
[32]
Vitamin B2 1.3 mg/day 1.1 mg/day Helps maintain the hair and skin healthy and critical for nerve functioning.
[32]
Vitamin B3 16 mg/day 14 mg/day Helps maintain the hair and skin healthy and critical for nerve functioning.
[32]
Vitamin B5 5 mg/day 5 mg/day Helps maintain the hair and skin healthy and critical for nerve functioning.
[32]
Vitamin B6 1.3 mg/day 1.3 mg/day Plays a role in sleep, appetite, and moods.
12 | P a g e Vitamin B9 400 µg/day 400 µg/day Helps prevent brain and spine
defects
[32] Vitamin B12 30 µg/day 30 µg/day Breaks down fatty acids. [32] Vitamin C 90 mg/day 75 mg/day Neutralizes unstable molecules that
can damage the cell and is an antioxidant.
[32]
Choline 550 mg/day 425 mg/day Helps transport and metabolize fats. [32] Vitamin D 31-70 µg/day 31-70 µg/day Maintains normal calcium and
phosphorus blood levels.
[32] Vitamin E 15 mg/day 15 mg/day Neutralizes unstable molecules that
can damage the cell and is an antioxidant
[32]
Vitamin K 120 µg/day 90 µg/day Helps activate proteins. It is essential for blood clotting.
[32] Carbohydrates
Glucose 70-99 mg/dL 70-99 mg/dL Supplies energy [33]
Lipids
Cholesterol <200 <200 Essential in making hormones and digest food.
[33] Triglyceride <150 mg/dL <150 mg/dL Provides energy and metabolism of
alcohol
[33] High-density
lipoprotein (HDL)
40-60 mg/dL 40-60 mg/dL Provides energy and metabolism of alcohol
[33] Low-density
lipoprotein (LDL)
<100 mg/dL <100 mg/dL Provides energy and metabolism of alcohol
[33]
2.1.4.4. Lipids
Lipids in the blood are either free or bound to another compound. These lipids can be influences by many factors such as diet, genetics, physical factors such as age and gender, health conditions, and lifestyle34.
No studies were found regarding the age of bloodstains and lipids. However, a study by Xiaoming Zhou35, did a five-year study on the effects of the climatic factors on the lipid levels in plasma, which can provide information about how lipids could react in a bloodstain over time. In five years' time, they investigated 47,270 people and did 82,526 checkups at the Shandong provincial hospital. All the blood samples acquired were collecting after overnight fasting. The chances are shown in Figure 6. The figure shows the difference between males and females and the temperatures. The results indicate that the main climatic factors, for example, humidity and precipitation do not affect the lipid levels significantly. Only air temperature could affect the lipid levels.
13 | P a g e Figure 6. Seasonal fluctuations of different lipids in blood plasma from men and women. A monthly average is shown of A (TC), B (TG), C (LDL), and D (HDL). The averaged air temperature. Reprinted with permission from Zhou, et al. 35.
There is a difference in the lipid levels between men and women. Men show a decrease in total cholesterol (0.35 mmol/L), LDL (0.18 mmol/L), and HDL (0.016 mmol/L), while TG increased with every 10 °C. For the woman, it was the TC values and HDL that noticeably decreased. For bloodstain analysis, this could mean that lipids could provide stable results over time while keeping in mind that air temperature can affect the results.
2.1.4.5. Amino acids
The human body needs twenty different amino acids for it to function correctly, although nine of these are labeled as essential (marked with * in Table 4), While there isn't a lot of research done regarding amino acid and determining the age of a bloodstain, it is still a field with a lot of opportunities, as amino acids are the building blocks for DNA, RNA, and proteins.
Table 4. Amino acids in the blood and their direct functions. * indicates the essential amino acids. The colors indicate the hydrophobicity of the amino acids; yellow= hydrophobic aliphatic; green= hydrophobic aromatic; gray= hydrophilic polar uncharged; purple= hydrophilic acidic; bleu= hydrophilic basic. n.d.= no data Adapted from [36-38]
Amino acid (Abbreviation) % Blood Concentration µmol/L Direct function Glycine (Gly)
3.8 245±16 Calcium influx through a glycine-gated channel in the cell membrane; purine and serine synthesis; synthesis of porphyrins; inhibitory neurotransmitter in the central nervous system
Alanine (Ala)
8.3 300±26 Inhibition of pyruvate kinase and hepatic autophagy; gluconeogenesis; transamination; glucose–alanine cycle *Valine 9.6 182.2±10.4 Synthesis of glutamine and alanine
14 | P a g e (Val)
*Leucine (Leu)
12.9 100.4±6.3 Regulation of protein turnover through cellular mTOR signaling and gene expression; activator of glutamate dehydrogenase flavor enhancer
*Isoleucine (Ile)
n.d. 55.5±3.4 Synthesis of glutamine and alanine Proline
(Pro)
4.9 177±9 Collagen structure and function; neurological function; osmoprotectant
*Methionine (Met)
1.2 22.3±1.8 Oxidant; independent risk factor for CVD; inhibition of NO synthesis
*Phenylalanine (Phe)
6.5 51±7 Activation of BH4 (a cofactor for NOS) synthesis; synthesis of tyrosine
Tyrosine (Tyr)
3.1 57.3±3.0 Protein phosphorylation, nitrosation *Tryptophan
(Trp)
0.2 n.d. Neurotransmitter; inhibiting the production of inflammatory cytokines and superoxide Serine
(Ser)
4.3 95.9±4.3 One-carbon unit metabolism; syntheses of cysteine, purine, pyrimidine; gluconeogenesis (particularly in ruminants); protein phosphorylation
*Threonine (Thr)
5.2 199.5±6.1 Synthesis of the mucin protein that is required for maintaining intestinal integrity and function; immune function; glycine synthesis
Cysteine (Cys)
2.3 44.3±6.9 Disulfide linkage in protein; transport of sulfur Asparagine
(Asn)
n.d. n.d. Cell metabolism and physiology; regulation of gene expression and immune function; ammonia detoxification Glutamine
(Gln)
n.d. n.d. Regulation of protein turnover through cellular mTOR signaling, gene expression, and immune function; syntheses of purine, pyrimidine, ornithine, citrulline, arginine, proline, and asparagine's; N reservoir; synthesis of NAD(P)
Aspartic Acid (Asp)
9.5 n.d. Coding for genetic information; gene expression; cell cycle and function; protein and uric acid synthesis; lymphocyte proliferation
Glutamic Acid (Glu)
8.3 67±18 Coding for genetic information; gene expression; cell cycle and function; protein and uric acid synthesis; lymphocyte proliferation
Arginine (Arg)
3.5 81.4±2.3 Activation of mTOR signaling; antioxidant; regulation of hormone secretion; allosteric; ammonia detoxification; regulation of gene expression; immune function; activation of BH4 synthesis; N reservoir; activation of NAG
synthase; methylation of proteins; deamination (formation of citrulline) of proteins
*Histidine (His)
6.9 72.6±3.6 Protein methylation; hemoglobin structure and function; antioxidative dipeptides; one-carbon unit metabolism *Lysine
(Lys)
9.4 140±14 Regulation of NO synthesis; antiviral activity (treatment of Herpes simplex); Protein methylation (e.g., trimethyl lysine in calmodulin), acetylation, ubiquitination, and O-linked glycosylation
2.1.4.6. Electrolytes
Electrolytes are mineral salts that are defined as the compounds that dissolve into cations and anions, meaning they have a charge and the ability to conduct electrical current39. The
electrolytes are present in body fluids as the fluid within the cells, in the space around the cells, and in blood. They help move nutrients into the cells, stabilizes the pH level in the body, helps maintain the water balance, and protects the body from infections. Loss of electrolytes occurs through sweating and urine, which can cause an imbalance40. With the right diet,
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everything stays in balance41. In the clinical laboratories, electrolytes such as sodium, calcium, potassium, and bicarbonate are often measured as a health screening. Table 5 shows the
electrolytes found in blood with their primary functions and the concentration range. The concentration range is based on the average "healthy" person. In literature, there are not many studies done regarding electrolytes and their aging in blood.
Table 5. Typical chemical composition of electrolytes in human blood plasma. The general function of all the electrolytes is described, and their concentration range in blood plasma is mentioned. These concentrations indicate the average healthy person.
Electrolyte Function Concentration range
(mmol/l)
Source
Sodium (Na+)
Regulates osmotic pressure gradient, between the cell’s interior and their surrounding environment
134.0 -145.0 [26, 42-44]
Potassium (K+)
Regulates the functioning of excitable tissues 3.0 - 5.5 [26, 42-44]
Calcium (Ca+)
Regulates the muscle contractions, nerve impulses and plays a role in the clotting of blood. Major cation in teeth
2.4 - 2.8 [26, 42-44]
Magnesium (Mg2+)
Effects muscle contractions, energy metabolism and many enzyme
0.75 - 1.3 [26, 42-44]
Bicarbonate (HCO3-)
It maintains the acid-base balance. It is part of the buddies buffer systems
24.0-29.0 [26, 42-44]
Phosphate (PO42-)
Important in bone and teeth structure 0.8 - 1.5 [26, 42-44]
Sulfate (SO4
2-)
Critical for protein matrix, detoxication of drugs, food, and metals. Helps prevent the blood from coagulating when it is being transferred through capillaries
0.5 - 1.0 [26, 42-44]
2.2. Blood serum
Previously in chapter 2.1.4, blood plasma and its properties were discussed. The papers reviewed often refer to blood plasma and blood serum as the same thing; however, in essence, they are not the same. The fundamental difference that blood plasma contains the protein fibrinogen, which helps to repair damaged tissue and form clots 45. As serum does not contain fibrinogen, it cannot clot. Due to the clotting factors of the plasma, it contains 10-20% more proteins than serum does, which loses these proteins while collecting the serum 46. Although plasma has more proteins than blood serum, it still contains enough proteins and other chemical components (gases, nutrients, nitrogenous wastes, hormones, and electrolytes) to provide substantial information regarding the research topic. Both plasma and serum are widely biological studies, clinical studies, and proteomics. The serum is often preferred when doing diagnostic testing, such as assays for cardiac troponin. At the same time, plasma is often used when studying diabetes, blood type testing clotting factors, and oral glucose
tolerance tests47-48. Both blood serum and plasma are used in the forensic science field, whole blood analysis, and bloodstains. In literature, the concentrations are often expressed as plasma concentration; however, often, serum is used.
Although the concentration of most compounds tends to be the same in serum and plasma, it is still important to research the difference between them 49. Zhonghao Yu 50 studied the difference between the metabolite profiles of Human plasma and serum. They
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analyzed the concentration of 163 metabolites in plasma and serum from 377 fasting people. This study showed that there is good stability in metabolite concentration in both the plasma and serum blood. Figure 7 shows the correlation between the measured metabolites in serum and blood plasma. Here the correlation coefficients (r) between the repeated measurements are plotted. The shapes represent the different metabolite groups studied. The r for plasma off all the metabolites was 0.83 and for serum 0.80. This shows that the reproducibility is
significantly better for plasma than for the serum samples, even though the difference in r is very small.
Figure 7. Correlation coefficients of the concentrations of the measured metabolites. Here the r values or serum are plotted against the two values of the metabolites in blood plasma. The shapes indicate different metabolites groups, and the colors represent the different subgroups of glycerophospholipids. This is an open-access article that permits unrestricted use. 50
This same paper also showed that serum and plasma metabolite concentrations have a high correlation (see Figure 8) the concentration of 85% of the metabolites in serum were significantly higher than in the plasma samples. Nine metabolites concentrations had a relative concentration difference of more than 20% with the amino acid Arg having the highest concentration difference of 50%. The partial least squares (PLS) analysis shows that the serum (in blue) and plasma (in red) samples are clearly separated (see Figure 9).
17 | P a g e Figure 8. Correlation between the concentrations of the measured metabolites in serum and plasma. The x-axis indicates the mean value of the relative concentration values. The y-axis indicates the r values. The shapes indicate different metabolites groups, and the colors represent the different subgroups of glycerophospholipids. This is an open-access article that permits unrestricted use 50.
Figure 9. PLS results. Here the scores of the first two PLS components are plotted against each other. The blue dots indicate the serum samples and the red plasma samples. This is an open-access article that permits unrestricted use 50.
When doing bloodstain analysis, it is important to know how stable blood is under different conditions, as environmental changes such as temperature and also time can affect the concentration and cause degradation. Flores, et al. 51 studied the stability of serum and blood plasma under different storage conditions. They stored the samples at 2,8 or -20°C for the duration of 15 to 30 days. The analytes they studied where: glucose, creatinine, uric acid, total and direct bilirubin. The Wilcoxon ranked-pairs test was used to assess the statistical differences between the samples. Table 6 shows the results acquired.
18 | P a g e Table 6. The concentration of the analytes in serum and in plasma, storage time, and temperature stored. * = the student T-test; **= The Wilcoxon ranked-pairs T-test; (p>0.05). n.d.= no data
Compound Median [interquartile range]
Mean difference % p-value source
Days Serum Plasma Serum Plasma Serum Plasma** [51]
Glucose 2- 8 °C (mmol/L) 0 4.86 [4.32-5.23] 5.02 [4.48-5.35] n.d. n.d. n.d. n.d. [51] 15 5.22 [4.66-5.73] 5.19 [4.70-5.62] 7.41 3.38 <0.001 <0.001 [51] 30 5.05 [4.36-5.33] 4.97 [4.33-5.40] 3.91 -0.99 <0.001 0.202 [51] Glucose -20°C (mmol/L) 0 4.86 [4.32-5.23] 5.02 [4.48-5.35] n.d. n.d. n.d. n.d. [51] 15 4.96 [4.37-5.23] 5.16 [4.69-5.44] 2.06 2.78 0.005 0.010 [51] 30 4.72 [4.14-5.11] 4.97 [4.44-5.26] 2.88 -0.99 <0.001 0.037 [51]
p-values serum vs plasma* 0.304 Creatinine 2- 8 °C (mmol/L) 0 64.1 [53.0-79.8] 63.7 [53.9-78.2] - - - - 15 75.1 [61.0-92.4] 71.6 [58.3-85.3] 17.2 12.4 <0.001 <0.001 [51] 30 78.2 [63.4-92.2] 72.1 [59.2-78.2] 22.0 13.2 <0.001 <0.001 [51] Creatinine -20°C (mmol/L) 0 64.1 [53.0-79.8] 67.7 [53.9-78.2] n.d. n.d. n.d. n.d. [51] 15 68.9 [56.6-84.2] 66.7 [54.6-83.1] 7.49 4.70 <0.001 <0.001 [51] 30 70.7 [59.0-84.2] 71.2 [57.2-86.9] 10.3 11.8 <0.001 <0.001 [51]
p-values serum vs plasma* 0.884 Uric acid 2- 8 °C (mmol/L) 0 0.24 [0.22-0.29] 0.23 [0.20-0.26] n.d. n.d. n.d. n.d. [51] 15 0.28 [0.24-0.32] 0.24 [0.22-0.30] 16.7 4.3 <0.001 <0.001 [51] 30 0.28 [0.24-0.32] 0.24 [0.22-0.29] 16.7 4.3 <0.001 <0.001 [51] Uric acid -20°C (mmol/L) 0 0.24 [0.22-0.29] 0.23 [0.20-0.26] n.d. n.d. n.d. n.d. [51] 15 0.25 [0.23-0.30] 0.24 [0.21-028] 4.16 4.3 <0.001 <0.001 [51] 30 0.26 [0.23-0.30] 0.25 [0.21-0.29] 8.33 8.7 <0.001 <0.001 [51]
p-values serum vs plasma* 0.212 Total bilirubin 2- 8 °C (mmol/L) 0 7.27 [5.09-12.7] 7.35 [4.92-12.1] n.d. n.d. n.d. n.d. [51] 15 4.79 [3.08-9.45] 3.42 [2.27-6.58] -34.1 -53.5 <0.001 <0.001 [51] 30 2.57 [1.33-4.66] 3.93 [1.88-6.84] -64.6 -46.5 <0.001 <0.001 [51] Total bilirubin -20°C (mmol/L) 0 7.27 [5.09-12.7] 7.35 [4.92-12.1] n.d. n.d. n.d. n.d. [51] 15 5.05 [3.38-11.4] 6.24 [4.02-11.4] -30.5 -15.1 <0.001 <0.001 [51] 30 1.45[0.68-4.53] 1.54 [0.86-3.51] -80.1 <0.001 <0.001 [51]
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-79.0
p-values serum vs plasma* 0.759 Direct bilirubin 2- 8 °C (mmol/L) 0 2.99 [2.57-5.13] 2.48 [2.01-3.81] n.d. n.d. n.d. n.d. [51] 15 2.05 [1.45-3.63] 1.37 [0.94-2.22] -31.4 -44.8 <0.001 <0.001 [51] 30 0.86 [0.34-1.71] 0.86 [0.51-1.24] -71.2 -65.3 <0.001 <0.001 [51] Direct bilirubin -20°C (mmol/L) 0 2.99 [2.57-5.13] 2.48 [2.01-3.81] n.d. n.d. n.d. n.d. [51] 15 2.65 [2.05-4.32] 2.39 [1.84-3.76] -11.4 -3.62 0.057 0.057 [51] 30 1.80 [0.98-2.74 1.80 [1.37-2.82] -39.8 -27.4 <0.001 <0.001 [51] p-values serum vs plasma* 0.078
Glucose, creatine, uric acid, and total bilirubin are unstable at both 15 days and 30 days when stored at -20°C; however, they are better preserved at this temperature.
Laboratories should freeze both the serum samples and plasma samples as soon as possible to achieve reproducible, stable results. This also means that the instable compounds analyzed are not suited for determining the age of bloodstains within a temperature range of -20 to 8 °C. the effects at higher temperatures have not been analyzed.
Another distinguishing factor of serum and blood plasma is the interaction of blood with materials and surfaces, as this plays a role in the final form and shape of the bloodstain. For example, bloodstains found in fabric (carpets, car seats, shirts, etc.) contain fewer
particles, as the fabric acts as a filter for the particles and even cells in blood52. Cell migration can get greatly affected depending on the tightness of the knit or weave in the fabric. Because of this, only serum can penetrate woven fabrics like yarn. RBCs may be found in between the yarns, but only serum will be absorbed into the yarn. Because of the clotting aspects of plasma, it is not often found in fabric samples that have aged52.
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3. Compounds being analyzed for bloodstain age determination
This chapter focusses on the different compounds/compounds classes that were proposed to determine the age of bloodstains. Figure 10 gives an overview of the compound classes analyzed for bloodstain age determination. The results indicate the proteins (enzyme proteins) are the most analyzed when determining the age of bloodstains, about 62% of the results. Nearly all proteins are enzymes, expect for RNA, which is an enzyme made of nucleic acid. Second are DNA and RNA, with 10 and 5% of the literature. DNA and RNA will bediscussed together as, in most cases they were also analyzed simultaneously.
Figure 10. Research articles used for this thesis clustered by compound class (n=42)
3.1. Proteins
As indicated in Figure 10, proteins are the most analyzed when determining the age of a bloodstain. In Table 7 is shown allTable 7. All proteins analyzed for bloodstain age determination. Of all the different proteins being analyzed, hemoglobin and hemoglobin derivatives are the most determined. Hemoglobin can be analyzed in both plasma, and red blood cells and are a very versatile compound to analyze. The presence of oxyhemoglobin in bloodstains is already visible from the start as it causes blood to change from a dark red color to a brownish color (as illustrated in Figure 4).
62% 5% 10% 2% 2% 5% 10% 2% 2% Protein DNA RNA Amino acids Hormones Drug RBC Alcohol Metabolites
21 | P a g e Table 7. All proteins analyzed for bloodstain age determination.
Compound Function Type of protein Age determination Source Phosphoglucomutase (PGM)
Transfers a phosphate group on an α-D-glucose
monomer
Enzyme Up to 20
months
[53-54]
Adenylate kinase (AK) Catalyzes the conversion of ADP to ATP and AMP
Enzyme One day to a
month
[53] Hemoglobin (Hb) Carries oxygen to the whole
body
Enzyme One day up to a year
[21, 55-67] Esterase D (ESD) Detoxification of
formaldehyde
Enzyme Up to 3 weeks [68] Globulins Liver function, blood
clotting, fighting infection
Plasma protein 30 days [69] Erythrocyte acid
phosphatase (EAP)
The activity of the prostate Enzyme Up to 30 days [70]
Haptoglobin (Hp) Binds free plasma HB Protein Up to 30 days [71] Immunoglobulin D (IgD) Protects the body from
infections
Antibody Up to 3 weeks [72] Group-specific
component (Gc)
Vitamin D-binding protein in plasma
Protein Up to 69 days [73] anti-HIV-1
IGB (immunoglobulin G)
Protects the body from infections An antibody, plasma protein Up to 6 months [74]
signal joint T-cell receptor rearrangement excision circles
(sjTRECs)
Marker Protein Up to days [75]
The enzyme PGM is known for transferring phosphate radicals on a glucose molecule from the 1-position to the 6-position in a reversed direction (see Figure 11). This process makes the PGM a key enzyme in gluconeogenesis and glycolysis 76.
Figure 11. Phosphoglucomutase (PGM) phosphate radical transferring reaction.
PGM shows to have a polymorphism in human blood, which shows potential for age determination 77. This was first discovered in 1964 by spencer et al. they discovered that there were three isoenzymes notated as PGM1, PGM2, and PGM 2-1. PGM is often used during forensic cases as it is present in many tissues, like bone-marrow and semen. The blood outside of the human body often starts with an increased activity, which deteriorates in time. The effect on the bloodstain sample depends on the conditions, such as temperature and bacterial
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growth. By electrophoresis focusing on fresh bloodstains, researchers were able to subtype PGM178. The method has been able to distinguish PGM in bloodstains up to 20 months successfully
Adenylate kinase (AK), also known as myokinase or ADK, is a phosphotransferase enzyme that has catalytic properties. AK transfers a phosphate from adenosine diphosphate (ADP) to another one. This produces adenosine triphosphate (ATP) and adenosine
monophosphate (AMP), as seen in Figure 12.
Figure 12. Reversible AK of two ADP molecules to generate ATP and AMP
AK can influence the local concentrations of ATP and ADP, with ATP being the most important one in terms of energy. The equilibrium of the AK reaction stays relatively stable in terms of pH and heat but is dependent on the magnesium concentration79.
In 1975 Parkin and Adams 68 analyzed the enzyme Esterase D (EsD). The EsD protein takes part in xenobiotic metabolism and is a carboxylesterase reaction. The EsD hydrolysis S-formylglutathione into formic acid and glutathione (see Figure 13), which is considered an esterase (thioesterase). The method has been able to distinguish AK for up to a month.
Figure 13. S-formylglutathione hydrolase of ESD into formic acid and glutathione.
Parkin and Adams 68 analyzed three phenotypes of EsD and named them EsD1, EsD2-1, and EsD2. The results showed that both EsD1 and EsD2 consist of three isoenzymes, while EsD2-1 consists of five. The samples stored at a low temperature of -20°C showed additional bands development. This same pattern was observed in bloodstains; however, the intensity of these additional bands faded as the stain aged. From the three phenotypes, EsD2 is easily distinguished with gel electrophoresis, but EsD1 and EsD2-1 are harder to identify. The method has been able to distinguish ESD in bloodstains up to 3 weeks old successfully.
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Grunbaum and Zajac 70 analyzed the enzyme Erythrocyte acid phosphatase (EAP) by using a florescent stain, to determine the age of blood, since EAP has five phenotypes that could be separated with electrophoretic methods. This method was able to successfully determine the age of blood up to 30 days (under ambient lab conditions).
3.2. DNA & RNA
DNA and RNA are the second most analyzed when it comes to bloodstain age determination. Here the DNA was extracted from the organic layer during the isolation of RNA from the same bloodstain. While DNA usually works best when the suspect can be found in the
database, it is still beneficial to link between the perpetrator and the victim. In 2005 Anderson, et al. 80 published a paper where they used real-time reverse PCR (polymerase chain reaction). In this paper, it is shown that over the course of 150 days, the ratio of 2 RNA molecules; 18 S rRNA and b-actin mRNA in human bloodstains exhibit a linear relationship in both female and male bloodstain (illustrated in Figure 14). The molecules 18 S rRNA and b-acting mRNA are both expressed in all cell types, thus making it very likely that they will be recovered at a crime scene. The paper stated that the cycle threshold (Ct) value for b-acting mRNA was significantly reduced over time; this meant that the relative ratio of 18S rRNA to b-actin mRNA increased as time progressed79.
Figure 14. One-way analysis of mean (ratio) by the age of ex vivo blood. It shows the change in RNA levels as a function of bloodstain age. Data represent the ratio of 18 S rRNA to b-actin mRNA as determined by real-time reverse transcriptase PCR. A model fitting the above sources of variability has a highly significant linear day effect (P < 0.0001) and has an adjusted R2 of 78.2%. Reprinted with permission from Anderson, et al. 80.
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While this method showed beneficial results for extracting DNA and RNA, it still required more study when it comes to the influence of environmental factors, as well as gender and ethnicity. In 2011 the same research group did a follow-up study, where they did a multivariate estimation using RT-PCR (reverse transcription-polymerase chain reaction) instead of PCR. This method combines the reverse transcription of RNA and DNA81. They analyze the age-related ratio of different RNA segments. The bloodstains analyzed had ex vivo ages of 0, 6,15, 30, and 90 days. The results of this report indicated that multivariate analysis could be used to distinguish samples of different ages. They used a nested analysis of variance to study the results of the population. This to determine both between and within donor variability. They also examined the experimental errors. Almost all factors were taken to be random, except for the age and sex of the sample. A hierarchical clustering algorithm, also known as Ward's method, was used to identify the cluster points of the RNA. This is done to demonstrate that clusters are being formed, hence a correlation with the age of blood. Figure 15 shows a dendrogram used to represent the agglomerative hierarchical clustering analysis performed. This is a three diagram that shows how the blood samples of the same age are clustered together while separated from different ages, which allows a more precise
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Figure 15. Dendrogram integrating results from all three amplicon combinations. Red lines are for bloodstains aged for zero-days, green for six days, blue for 30 days, and orange for 90 days. Reprinted with permission from Anderson, et al. 81.
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This method can differentiate the 90-day old bloodstain from the 30 days old. While this proved to be a good thing, this method is still flawed as the authors stated that the found ratios are ineffective and not accurate enough to measure the age of bloodstains. This has to do with the similar decay of different markers analyzed.
Like their previous study, the effect of the environment was not analyzed. Alshehhi and Haddrill 82 studied the degradation of RNA markers in 2017 with a follow-up study in 2019. The RNA markers being investigated were a mix of pure and mixed body fluid samples measured for bloodstain ages up to 60 days. The levels of the blood-specific markers, HBA (human hemoglobin), HBB (human β-globin gene), and MiR16, together with the reference genes 18S and U2, showed no significant differences when compared. While all these studies seem promising, they are still lacking, and further research and validation are needed.
3.2.1. DNA methylation assay
A few articles used a DNA methylation assay. DNA methylation is a biological mechanism that transfers a methyl group onto cytosine to form 5-methylcytosine (illustrated in Figure 16). DNA methyltransferase (DNMT) enzymes do this process. The majority of DNA methylation occurs on cytosines followed by guanine nucleotide, also known as CpG sites. Although DNA methylation appears to be relatively stable in cells, changes in the histone pattern can occur. This can happen rapidly during the cell cycle. Despite this phenomenon happening in the cell, many studies indicate that the linking on the position is indeed connected83.
Figure 16. DNA methylation: transfer from a methyl group onto the C5 position to form 5-Methylcytosine.
The studies about DNA methylation stated that the aging process is linked to epigenetic modifications. Methylation changes at specific oligodeoxynucleotides (CpG) sites84-85. Some CpG sites show a linear correlation with aging, that can be used as a
biomarker to predict the age of bloodstains. Peng, et al. 86 used the pyrosequencing approach to screen CpG sites for age determination purposes. This approach is based on the sequencing by synthesis principle, where the sequencing is done by detecting the nucleotide. The
methylation levels of 99 blood samples were analyzed, and the time-series of the bloodstains were converted into gauze and on Flinders Technology Association (FTA) cards. The results indicate that the DNA methylation levels at all CpG sites show a high correlation between the different analyzed groups (FTA results illustrated in Figure 17). The results were acquired under room temperature conditions (25 °C), which shows the high potential of this method.
27 | P a g e Figure 17. Methylation levels of each CpG site from the 30 blood samples and the time-series on FTA (Flinders Technology Association) cards. The different colors represent different samples groups: (color red: blood-0 h, blue: FTA-24 h, green: FTA-7d, purple: FTA-30d, orange: FTA-3 m). P-values less than 0.05, 0.005 and 0.0005 are represented in *, ** and ***, respectively. Reprinted with permission from Peng, et al. 86
3.3. Amino acids determination
Out of the twenty amino acids present in the human body, only one of them has been used to determine the age of bloodstains: aspartic acid. Arany and Ohtani 87 provided an introductory study based on the aspartic acid racemization (AAR) present in aging bloodstains. Amino acids exist in two enantiomeric forms, de D, and the L form. In the human body, all amino acids are initially in the L enantiomeric form. Racemization converts the L-form into a mixture of D- and L-form. Due to the slow conversation rate of this process, it can be used to estimate the age of a protein88-89. The AAR procedure performed by Arany and Ohtani 87 involved dissolving the amino acids present, followed by a centrifuge step and lastly being analyzed by GC, which is a destructive method. However, this process is very commonly used in medical applications to assess the aging of proteins. While the results of this study initially appeared to show a linear correlation, it was found that it is profoundly affected by the
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environmental temperature. Its seen in Figure 18 that d-aspartic acid has a direct relationship to a bloodstain, but this increases at different temperatures, as seen in graphic a.
Figure 18. (a) Heating experiments were performed to investigate Asx racemization in a standard bloodstain (obtained from a healthy volunteer). The D-Asx content of the standard bloodstain showed a linear increase at each temperature: 90 °C Reprinted with permission from Arany and Ohtani 87
Now, this method is not developed enough as it gets influenced a lot by environmental parameters. Although every amino acid, for example, leucine, that is the highest percentage in blood or reacts differently to AAR, it would still be a good field to analyze the other ones like glutamic acid as it has similar qualifications to aspartic acid (Table 4).
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4. Analytical techniques for determining the age of bloodstains
Several techniques have been used in the past for obtaining chemical information regarding the age of fingerprints. Figure 19 shows analytical method classes found in the literature used for bloodstain age analysis. In Table 8, the employed analytical techniques are summarized by the age of the bloodstain analyzed, and the advantages and disadvantages of the methods. Based on the results, electrophoretic methods and spectroscopy are the two most used method classes. In the next chapter, the more stand out and interesting methods will be discussed.Figure 19. Research articles by analytical method used for this thesis. (n=42)
Table 8. Overview of used analytical methods for analyzing the age of bloodstains, with the pros and cons of the methods.
Method Bloodstain age determination
Advantages of the method
Disadvantages Source
Electrophoresis Up to 24 months Possibility to alter the gel Commercial
Give Hb levels
Time-consuming
Inaccurate quantification Poor precision and accuracy compared to other techniques [53-54, 68, 70-71] Immunoelectrop horetic
Up to 1 year High resolution due to applied field and pH gradient
Minimal band spreading
High voltage power Carries generally in high concentrations
[69, 73]
Isoelectric focusing (IEF)
Up to 7 months Versatile identification Accurate
Low cost
Toxicity
Limited sample analysis Precision is poor [90] Laser-induced fluorescence capillary electrophoresis Up to 6 months Fluorescence Speed Resolution Efficiency, sensitivity Instability laser
Excitation range limited Derivation required No standardized methods
30 | P a g e Spectroscopy methods Up to 4 months [55] Uv-vis (Ultraviolet-visible spectroscopy)
Up to 1 year High sensitivity Small sample volume required
Limited wavelength range Lack of sensitivity and selectivity
[21, 62]
Atomic force spectroscopy (AFM)
Up to 31 days Easy preparation Fast
No problem surface pollution
High contact pressure Limited vertical range Data not independent op tip
[63, 92-93]
Raman spectroscopy
up to a year Little sample prep Less sensitive to temperature changes High specificity Organic and inorganic material detection
Prone to interference for proteins like hemoglobin Unstable laser wavelength Long collection time Low sensitivity [64-65] Near-infrared spectroscopy Up to 107 days Non-destructive No chemical waste High scan speed Minimal sample prep High resolution
Sensitive to interferences like temperature, pressure changes
Lack of sensitivity for low protein concentration [6, 67, 94] Electron paramagnetic spectroscopy (EPR)
Up to 2 months Small sample size Detection time
Complicated spectra Hard to do very small blood samples Sensitivity paramagnetic compounds [56, 60] Scanning electron microscopy (SEM)
Up to 18 months High resolution Wide range of magnifications
Not real-time
Requires additional sample prep [59] Chromatography Reversed-phase- HPLC Up to 52 weeks Speed High resolution Sensitivity Accuracy Automation Complexity [57-58] Gas-liquid chromatography (GLC) Up to 6 months Sensitive
Suitable for volatile organics Destructive Limited to volatile compounds [95] Gas chromatography (GC-MS)
Up to 20 years High sensitivity Good dynamic range Reproducible Short run time
Destructive Limited to volatile compounds
[4, 87, 96]
HPLC-MS/MS Up to 21 days High specificity High sensitivity Wide dynamic range Less sample prep
Destructive Limited to volatile compounds [4, 97] PCR Up to 6 months Up to 150 days Up to a year High sensitivity Easy to use High level of repeatability and reproducibility
Setting and running Potential lower specificity
[75, 80-81, 91, 98]
31 | P a g e Single Radial
immunodiffusion (SRID)
Up to 55 days Specific and sensitive results
Good for large amounts of antigens
Large sample needed [72]
ELISA enzyme-linked immunosorbent assay Up to 90 days Robust Easy to use High level of repeatability and reproducibility Limited antigen information [74]
EpiTYPER Up to 3 months DNA Input low Large amount
Intermediate methylation levels expected [86] Smartphone Samsung Up to 3 days Portable Cheap Fast, easy Lighting problems Homogenous conditions [66] 4.1. Electrophoretic techniques
Electrophoretic techniques are one of the early pioneer's methods when it comes to bloodstain age determination. One of the first articles being from 1970, where Rothwell 53 analyzed the effect of storage on the activity of phosphoglucomutase and adenylate kinase enzymes in blood samples and stains. They used a thin layer of starch gel.
After the band became visible, they were assets on an arbitrary scale, which is seen in in Figure 20, where:
0. means that there was no visible activity.
1. means that there was some faint blue coloration, but no discrete bands where formed. In 0 and 1, the characterization of phosphoglucomutase and adenylate kinase is not possible. For 2 and 3, it is.
2. Means that there where discrete line but were not intense. 3. This means that the lines were discrete and intense.
