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Chapter 10: Serology and Bloodstain Pattern Analysis
10.1: Introduction
Serology – a major component of crime scene processing and analysis.
It also plays a large role in the processing of items of evidence in the laboratory, presumptively identifying blood, semen, saliva, and urine before further analysis.
It is a conservative method, in that, while the stain may be identified, only a portion of that stain is further analyzed and the identified remainder is reserved for future testing.
Proteomics – the study of proteome; covers all the proteins in any given cell, their various forms and modifications, interactions, structure, the higher order complexes they form, and pretty much everything that happens “post-genome.”
Proteome – the set of proteins coded in the genetic makeup of an individual, the genome.
10.2: Collection of Body Fluids
Serological analysis, like other forensic analyzes, has two types of tests.
Presumptive test – highly sensitive to but not specific for a particular substance.
The idea of presumptive tests is that some false positive results are acceptable as long as no false negative results are obtained. False-negative results are possible, of course. The presumptive positive results can be more specifically tested with the second type of test.
Confirmatory Test – which tests positive for the substance in question and only that substance.
It lacks sensitivity, in that a relatively large amount of the substance must be available for the test to be positive, but a few false negatives are acceptable as long as no false positives are obtained.
10.3: The Major Body Fluids
Blood
Blood is a tissue, composed of several types of cells in a matrix called plasma.
Plasma consists of about 90% water and 10% of a long list of other substances (7% protein, 3% urea, amino acids, carbohydrates, organic acids, fats, steroid hormones, and other inorganic ions).
Three Types of Cells
Erythrocytes – RBC; are legion in the blood—roughly 5 million per milliliter of blood! The purpose of these 6–8μm diameter cells is to transport oxygen and carbon dioxide throughout the body via the circulatory system; this is accomplished by hemoglobin.
Hemoglobin – the respiratory pigment of many animals, is a conjugated protein consisting of four polypeptides, each of which contains a heme group.
The heme group – contain iron and have the ability to bind with oxygen; this association is reversible, allowing for respiration.
Erythrocytes are produced in the bone marrow and have about a 4-month life span. They discard their nuclei as they mature and, therefore, contain no DNA.
Leukocytes – WBC; are active in the immune system, about 10–15,000 per milliliter of blood.
Neutrophils – are part of the first line of defense and offer up a complicated response to invaders: the immune response.
Lymphocytes – produced in the bone marrow and the thymus gland, to engender the immune response. Lymphocytes produce antibodies — which are protein molecules that can bind to foreign molecules. Any foreign molecule that induces antibody formation is called an antigen.
Macrophages support the immune response. Once bacteria or damaged platelets are identified, macrophages swarm the area and consume the offending materials until they eat themselves to death.
Platelets – are only fragments of cells and contain no nuclei; they number around 15–300,000 per milliliter of blood. Platelets are involved in the clotting process.
Blood group – a class of antigens produced by allelic genes at one or more loci and inherited independently of other genes.
ABO blood group – discovered in 1900 by Karl Landsteiner.
The letters refer to the antigens on the surface of the red blood cells; corresponding antibodies, anti-A, and anti-B are present in the plasma.
A person with Type B blood will have anti-A antibodies in his or her plasma. If the plasma is mixed with Type A blood, the cells will agglutinate, or clump together, from the reaction of the Type A antigens and the anti-A antibodies.
Blood Testing
Phenolphthalein, benzidine, leucomalachite green, and tetramethylbenzidine (TMB) are – the most commonly used catalytic color tests.
Luminol – reacts in the presence of hemoglobin, much like phenolphthalein, when an oxidizer is applied. It is very sensitive to hemoglobin and will detect blood in dilutions of 1 in 5,000,000.
Fluorescein — the chemical that is used to check for the presence of blood and is prepared much in the same way as luminol except that the commercial preparation contains a thickener.
Takayama Test – also known as the hemochromogen test; is performed by taking a small sample of the presumptive stain and placing it under a coverslip.
Ouchterlony test – a common diffusion reaction test; is based on an antibody–antigen reaction between human blood and human antiserum.
Human antiserum – typically produced by injecting rabbits with human blood.
Electrophoresis methods – based on the diffusion of antibodies and antigens on an electrically charged gel-coated plate.
Semen
Semen – a complex gelatinous mixture of cells, amino acids, sugars, salts, ions, and other materials produced by postpubescent males and is ejaculated following sexual stimulation.
Spermatozoa – a specialized structure approximately 55μm in length with a head containing DNA and a tail that wiggles, or flagellates, to produce movement
Acid phosphatase – a common enzyme in nature that occurs at a very high level in semen.
Brentamine Fast Blue B – most common semen test; applied to the sample on an alpha-naphthyl phosphate substrate.
Christmas tree stain – turns the tip of the sperm’s head pink, the bottom of the head dark red, the middle portion blue, and the tail yellowish-green; skin cells stain green to blue-green and are easily distinguished.
Enzyme-linked immunosorbent assay (ELISA) – used to detect p30 at levels as low as 0.005 ng/mL.
Depending on the crime, it may be useful to determine the time since intercourse (TSI) to assist in the sequence of events.
Motile sperm can survive in the vagina for about 3h, ranging from 1 to 8h; they survive longer in the cervix, up to several days in some cases.
Saliva
Bite marks, licked adhesives (like envelopes and stamps), eating and drinking surfaces, or even expectoration (spitting) can yield important DNA evidence. Saliva stains may be difficult to see, and detection can be tricky.
Radial diffusion test – an old test used to be employed to confirm the presence of saliva, but now the sample is considered presumptively positive for saliva and simply sent on for DNA analysis.
Urine
Urine – the excreted fluid and waste products filtered by the kidneys, can be presumptively tested for through the presence of urea or creatine.
When heated, a urine stain gives off a characteristic odor that everyone is familiar with.
10.4: Bloodstain Pattern Analysis
Bloodstain pattern analysis (BPA) – the analysis and interpretation of the dispersion, shape characteristics, volume, pattern, number, and relationship of bloodstains at a crime scene to reconstruct a process of events.
Passive Bloodstains – include clots, drops, flows, and pooling.
Transfer bloodstains – include wipes, swipes, pattern transfers, and general contact bloodstains.
Projected / Impact Bloodstains – spatters, splashes, cast-off stains, and arterial spurts or gushes.
Wipe Stain – created when an object moves through a pre-existing bloodstain.
Spatter – a technical term in BPA that describes a stain that results from blood hitting a target.
Forward spatter – results when blood droplets are projected away from the item creating the impact, such as a hammer.
Back Spatter – caused by droplets being projected toward the item.
Cast-off stains – the result of blood being flung or projected from a bloody object in motion or one that stops suddenly.
Arterial spurts/gushes – can vary due to the pumping action and variable pressure of the blood as it exits the wound, producing a zig-zag, up-and-down pattern
Fly spots – stains resulting from fly activity—and may mimic other relevant BPA patterns.
Voids – an indicator that some secondary object came between a blood spatter and the final target; this leaves an outline or “shadow” on the final target.
As a stain dries, the edges and borders dry first due to surface effects. If the bloodstain is wiped, these dried areas, called skeletonized stains, remain behind.
Directionality – demonstrates the vector of a droplet when it hits the target; the tail points in the direction of travel
Direction Angle – the angle between the long axis of the stain and a standard reference point, usually 0° vertical.
Satellite droplets – are small amounts of blood that detach from the parent stain and “splash” onto a surface.
Determining the point of origin of one or more bloodstains is central to the reconstruction of a blood-related event.
10.5: Documenting Bloodstains at the Scene
The documentation of bloodstains is painstaking work but crucial to a successful reconstruction.
Here are the following recommended photographic guidelines:
Document the entire scene as discovered, including “establishing” photographs.
Photograph pattern transfers, pools, and other fragile patterns first.
Document patterns with “establishing” photographs that show the pattern’s relationship to landmarks or other items of evidence.
Take macro and close-up photographs; include a scale in every photograph.
When reconstructing point-of-origin, document individual stains used in the reconstruction.
Chapter 10: Serology and Bloodstain Pattern Analysis
10.1: Introduction
Serology – a major component of crime scene processing and analysis.
It also plays a large role in the processing of items of evidence in the laboratory, presumptively identifying blood, semen, saliva, and urine before further analysis.
It is a conservative method, in that, while the stain may be identified, only a portion of that stain is further analyzed and the identified remainder is reserved for future testing.
Proteomics – the study of proteome; covers all the proteins in any given cell, their various forms and modifications, interactions, structure, the higher order complexes they form, and pretty much everything that happens “post-genome.”
Proteome – the set of proteins coded in the genetic makeup of an individual, the genome.
10.2: Collection of Body Fluids
Serological analysis, like other forensic analyzes, has two types of tests.
Presumptive test – highly sensitive to but not specific for a particular substance.
The idea of presumptive tests is that some false positive results are acceptable as long as no false negative results are obtained. False-negative results are possible, of course. The presumptive positive results can be more specifically tested with the second type of test.
Confirmatory Test – which tests positive for the substance in question and only that substance.
It lacks sensitivity, in that a relatively large amount of the substance must be available for the test to be positive, but a few false negatives are acceptable as long as no false positives are obtained.
10.3: The Major Body Fluids
Blood
Blood is a tissue, composed of several types of cells in a matrix called plasma.
Plasma consists of about 90% water and 10% of a long list of other substances (7% protein, 3% urea, amino acids, carbohydrates, organic acids, fats, steroid hormones, and other inorganic ions).
Three Types of Cells
Erythrocytes – RBC; are legion in the blood—roughly 5 million per milliliter of blood! The purpose of these 6–8μm diameter cells is to transport oxygen and carbon dioxide throughout the body via the circulatory system; this is accomplished by hemoglobin.
Hemoglobin – the respiratory pigment of many animals, is a conjugated protein consisting of four polypeptides, each of which contains a heme group.
The heme group – contain iron and have the ability to bind with oxygen; this association is reversible, allowing for respiration.
Erythrocytes are produced in the bone marrow and have about a 4-month life span. They discard their nuclei as they mature and, therefore, contain no DNA.
Leukocytes – WBC; are active in the immune system, about 10–15,000 per milliliter of blood.
Neutrophils – are part of the first line of defense and offer up a complicated response to invaders: the immune response.
Lymphocytes – produced in the bone marrow and the thymus gland, to engender the immune response. Lymphocytes produce antibodies — which are protein molecules that can bind to foreign molecules. Any foreign molecule that induces antibody formation is called an antigen.
Macrophages support the immune response. Once bacteria or damaged platelets are identified, macrophages swarm the area and consume the offending materials until they eat themselves to death.
Platelets – are only fragments of cells and contain no nuclei; they number around 15–300,000 per milliliter of blood. Platelets are involved in the clotting process.
Blood group – a class of antigens produced by allelic genes at one or more loci and inherited independently of other genes.
ABO blood group – discovered in 1900 by Karl Landsteiner.
The letters refer to the antigens on the surface of the red blood cells; corresponding antibodies, anti-A, and anti-B are present in the plasma.
A person with Type B blood will have anti-A antibodies in his or her plasma. If the plasma is mixed with Type A blood, the cells will agglutinate, or clump together, from the reaction of the Type A antigens and the anti-A antibodies.
Blood Testing
Phenolphthalein, benzidine, leucomalachite green, and tetramethylbenzidine (TMB) are – the most commonly used catalytic color tests.
Luminol – reacts in the presence of hemoglobin, much like phenolphthalein, when an oxidizer is applied. It is very sensitive to hemoglobin and will detect blood in dilutions of 1 in 5,000,000.
Fluorescein — the chemical that is used to check for the presence of blood and is prepared much in the same way as luminol except that the commercial preparation contains a thickener.
Takayama Test – also known as the hemochromogen test; is performed by taking a small sample of the presumptive stain and placing it under a coverslip.
Ouchterlony test – a common diffusion reaction test; is based on an antibody–antigen reaction between human blood and human antiserum.
Human antiserum – typically produced by injecting rabbits with human blood.
Electrophoresis methods – based on the diffusion of antibodies and antigens on an electrically charged gel-coated plate.
Semen
Semen – a complex gelatinous mixture of cells, amino acids, sugars, salts, ions, and other materials produced by postpubescent males and is ejaculated following sexual stimulation.
Spermatozoa – a specialized structure approximately 55μm in length with a head containing DNA and a tail that wiggles, or flagellates, to produce movement
Acid phosphatase – a common enzyme in nature that occurs at a very high level in semen.
Brentamine Fast Blue B – most common semen test; applied to the sample on an alpha-naphthyl phosphate substrate.
Christmas tree stain – turns the tip of the sperm’s head pink, the bottom of the head dark red, the middle portion blue, and the tail yellowish-green; skin cells stain green to blue-green and are easily distinguished.
Enzyme-linked immunosorbent assay (ELISA) – used to detect p30 at levels as low as 0.005 ng/mL.
Depending on the crime, it may be useful to determine the time since intercourse (TSI) to assist in the sequence of events.
Motile sperm can survive in the vagina for about 3h, ranging from 1 to 8h; they survive longer in the cervix, up to several days in some cases.
Saliva
Bite marks, licked adhesives (like envelopes and stamps), eating and drinking surfaces, or even expectoration (spitting) can yield important DNA evidence. Saliva stains may be difficult to see, and detection can be tricky.
Radial diffusion test – an old test used to be employed to confirm the presence of saliva, but now the sample is considered presumptively positive for saliva and simply sent on for DNA analysis.
Urine
Urine – the excreted fluid and waste products filtered by the kidneys, can be presumptively tested for through the presence of urea or creatine.
When heated, a urine stain gives off a characteristic odor that everyone is familiar with.
10.4: Bloodstain Pattern Analysis
Bloodstain pattern analysis (BPA) – the analysis and interpretation of the dispersion, shape characteristics, volume, pattern, number, and relationship of bloodstains at a crime scene to reconstruct a process of events.
Passive Bloodstains – include clots, drops, flows, and pooling.
Transfer bloodstains – include wipes, swipes, pattern transfers, and general contact bloodstains.
Projected / Impact Bloodstains – spatters, splashes, cast-off stains, and arterial spurts or gushes.
Wipe Stain – created when an object moves through a pre-existing bloodstain.
Spatter – a technical term in BPA that describes a stain that results from blood hitting a target.
Forward spatter – results when blood droplets are projected away from the item creating the impact, such as a hammer.
Back Spatter – caused by droplets being projected toward the item.
Cast-off stains – the result of blood being flung or projected from a bloody object in motion or one that stops suddenly.
Arterial spurts/gushes – can vary due to the pumping action and variable pressure of the blood as it exits the wound, producing a zig-zag, up-and-down pattern
Fly spots – stains resulting from fly activity—and may mimic other relevant BPA patterns.
Voids – an indicator that some secondary object came between a blood spatter and the final target; this leaves an outline or “shadow” on the final target.
As a stain dries, the edges and borders dry first due to surface effects. If the bloodstain is wiped, these dried areas, called skeletonized stains, remain behind.
Directionality – demonstrates the vector of a droplet when it hits the target; the tail points in the direction of travel
Direction Angle – the angle between the long axis of the stain and a standard reference point, usually 0° vertical.
Satellite droplets – are small amounts of blood that detach from the parent stain and “splash” onto a surface.
Determining the point of origin of one or more bloodstains is central to the reconstruction of a blood-related event.
10.5: Documenting Bloodstains at the Scene
The documentation of bloodstains is painstaking work but crucial to a successful reconstruction.
Here are the following recommended photographic guidelines:
Document the entire scene as discovered, including “establishing” photographs.
Photograph pattern transfers, pools, and other fragile patterns first.
Document patterns with “establishing” photographs that show the pattern’s relationship to landmarks or other items of evidence.
Take macro and close-up photographs; include a scale in every photograph.
When reconstructing point-of-origin, document individual stains used in the reconstruction.
NoteStudied by 22 peopleNoteStudied by 13 peopleNoteStudied by 7 peopleNoteStudied by 39 peopleNoteStudied by 5 peopleNoteStudied by 14 people