Understanding Serology and Biological Evidence | Criminal Defense and DNA | Fresno, Ca (2)

Understanding Serology and Biological Evidence | Criminal Defense and DNA | Fresno, Ca (2)

Searching a Crime Scene for Blood and Other Body Fluids

Serology is the detection, identification, and typing of body tissues, in native form or in the form of stains or residues left at a crime scene. The ability to critically observe the scene and good common sense are two main characteristics of a good crime scene investigator, who has the responsibility of noting anything unusual at the scene and documenting those findings. Back in the laboratory, the forensic analyst plays the same role in collecting, recording, preserving, and analyzing evidence from those items recovered.

Biological evidence is most informative in cases when the suspect and victim come into close contact or struggle with each other. In stabbing and beating incidents, the close proximity of the assailant to the victim can result in the transfer of blood between the two. During assailant-victim struggles, skin and blood may be transferred to the victim's fingernails.

Throughout this section we consistently refer to the processes of locating and processing of bloodstains, however the principles addressed are equally applicable to locating, collecting, and preserving all types of biological fluids and stains.

When processing a crime scene, the most fragile evidence is collected first, and then more obvious evidentiary items are identified next. In some case scenarios, special techniques may be used to locate some of the "hidden" evidence. It is not unusual for a perpetrator to try to conceal or hide evidence by cleaning up blood-contaminated areas. However, blood is actually very difficult to remove from a scene completely. Since blood is a fluid, it can flow between floorboards, under baseboards, and into carpeting. In such cases, it may be more useful to conduct searches using high intensity light sources that can aid in the visualization of bloodstains and other body fluids, thereby minimizing the loss of evidence at the scene.

Light Sources

Since the use of visible light may be insufficient to reveal or locate some types of biological evidence, other types of light may be necessary.  Lasers and alternative light sources may be used to reveal hidden or undetected biological fluids and evidence. Alternative light sources (ALS) are also routinely used for processing fingerprints, questioned documents, shoeprints and detection, trace evidence (hair, fibers, glass fragments), gunshot residues, bite marks, bruises and human bone fragments. There isn't a single laser or light source that can be used universally for all forensic applications, so a number of factors must be considered when choosing a high intensity light for a given forensic application. These factors include:

  • Cost
  • Color of light or wavelength
  • Light intensity or power output
  • Portability
  • Voltage requirements
  • Safety
  • Range of applications

To enhance a stain in contrast to its background, light must shine on the stain in a way that little or no reflection occurs. The wavelength of light used must be a wavelength opposite to the color of the stain. The ALS is composed of the light either a laser or an incandescent bulb, and a filter or a combination of filters that screen out all wavelengths except the selected wavelength that is delivered to the evidence. Various filters enable the visualization of fluorescence emitted by the stain or the biological material. The majority of organic materials can fluoresce (absorb light at one wavelength and emit it again at a longer wavelength) semen, urine and vaginal secretions all fluoresce white using an orange filter. In fact most protein rich stains will also fluoresce white. Under UV light, blood doesn't fluoresce but shows up as a black/dark area, which can be prominent if the rest of the item fluoresces.  The detection of blood in obscure areas can often be carried out with a high intensity light, enabling samples to be located, obtained and taken to the laboratory for further testing. Light sources can also be used to provide oblique (side) lighting, which is an excellent means of finding trace evidence and other small items of interest especially on large items such as bedding, comforters and carpets. The use of these light sources may require specialized photographic techniques to record the evidence.

Luminol

In some crime scenarios, stain visualization enhancing chemicals might be used to locate or identify areas of suspected blood staining. The most popular chemical used is Luminol. When Luminol is applied to bloodstains the stains glow in the dark, even if the stain is very dilute or if the blood is present in trace amounts. However, Luminol use is associated with a number of disadvantages and should only ever be used as a last resort.

For genetic analysis to be carried out on a sample, enough stain must be recovered to allow confirmatory tests to be conducted. This amount of blood is usually sufficient to be visible to the naked eye. If a stain is not visible without Luminol treatment there is probably insufficient sample for further confirmatory or genetic testing. Complete darkness is also necessary to view the luminescence created by a positive Luminol reaction, which can be difficult to achieve at some crime scenes. The requirement for darkness can hamper processing and photography of any potential evidence revealed. Due to the chemical make-up of this reagent it is also considered to be one of the most carcinogenic reagents used by forensic investigators. These disadvantages associated with Luminol use probably preclude its more routine use. Like many other chemical tests that react with bodily fluids Luminol can give false positive reactions. Luminol is a presumptive test for blood. A presumptive test is used to indicate the possible presence of a substance, but is not a specific test for that substance. A test that can unequivocally identify a substance is called a confirmatory test. Luminol cannot be considered confirmatory since the agent also reacts with copper and iron compounds, cobalt ions, potassium permanganate, bleach and plant peroxidases.

Leucocrystal Violet (LCV)

LCV is a reagent that reacts with heme in blood to produce a violet color. Like luminol it can be used to identify latent blood prints and to enhance visible blood stain patterns. This reagent is usually used on porous surfaces and has been successfully used to enhance bloody footwear impressions and bloody foot and fingerprints.

Amido Black

Amido black is a highly sensitive stain that produces a light to medium blue color when it reacts with blood proteins. Results with amido black can be difficult to discern on multi colored surfaces and porous surfaces, but it works very well on non-porous surfaces to enhance latent fingerprints and other impressions in blood.

Collection and Preservation of Biological Evidence

During the evidence collection phase of a crime scene investigation, the events that occurred at the scene are not always immediately obvious. Therefore it is prudent for the scene examiner to collect and record evidence in a way that the integrity of the evidence is preserved in case further examination of the evidence is required at a later date. Crime scene investigators also need to determine what evidence needs to be forwarded to specialty sections of the laboratory for detailed analysis (chemistry, trace evidence, biology/serology). This may result in several pieces of evidence being examined sequentially by each specialty section, requiring more than one type of scientific analysis to fully process the evidence.

As biological samples are collected at the scene, it is important to remember the facts you are trying to establish within the investigation; the evidence collected must provide the most information. With bloodstains it's important to collect representative samples from areas away from the "action", or from bloodstains and spatters that appear different from the majority of other stains present, since these stains may provide the most information. Bloodstains leading away from a scene may have come from an injured suspect or assailant and may provide information leading to their identification.

Analysis of biological evidence requires the comparison of the evidence with standard reference samples obtained from the victim and suspect.

Packaging

Most items of evidence will be collected and packaged in clean, unused paper containers such as packets, envelopes, and bags. As a rule of thumb never package in plastic. However as we discuss this further we'll address some situations where this rule doesn't necessarily apply for certain types of evidence. Before transporting or packaging items of evidence, the investigator should carefully examine the items to recover any loose trace evidence (hairs, fibers, paint chips, etc.) that may be lost during transport to the laboratory. Loose evidence should be collected in a paper packet and placed in an envelope, labeled with the appropriate information, such as a description of the item and the source of the trace evidence, to maintain chain of custody. Each package should be sealed with tamper-proof tape (never stapled) and the seal initialed by the person packaging the evidence. In the laboratory the analyst receiving the evidence will check to make sure this seal is still intact and hasn't been tampered with.

Packaging of Dried Stains

If it's possible to transport the entire stained item to the laboratory it should be packed in a paper bag or envelope if possible. By having access to the entire item the analyst will be able to decide which areas of the staining should be further tested, while decreasing the risk of contamination or stain dilution by using other collection methods. Bulkier items are more difficult to handle. If the stained item is extremely large or not easily transported, samples can be removed from the item in a variety of ways. It is also important to obtain samples of unstained areas to use as control samples. Samples can be obtained from large items by cutting out a portion of a stain. These samples should then be packaged in paper envelopes and submitted to the laboratory. Some stains can be recovered from surfaces using fingerprint tape. The sticky tape is rubbed over the stain then lifted and placed on a vinyl or acetate sheet that is then packaged and stored in an envelope. Alternatively, a cotton swab moistened with water can be rubbed over the stained area and some of the stain will be absorbed onto the swab. Dried and crusted stains can be obtained by scraping the stain into separate paper packets, which are then labeled and further packaged in a paper envelope.

Packaging of Wet Stains

Wet stains, especially those from hard surfaces, may be collected by absorbing the stain onto long cotton threads, small squares of cotton gauze or cotton tipped swabs. Although an effective means of collection for wet stains, when this technique is used to collect dried stains the cotton must be wetted which may result in stain dilution or contamination

Although the general rule is no packaging in plastic, large moist or wet biological evidence may be collected in clean, unused plastic containers at the scene to prevent contamination of other evidence prior to transportation to an evidence receiving area, or the laboratory. However, the storage time in sealed plastic must be absolutely minimal (less than two hours) to avoid bacterial degradation and sample putrefaction, which can destroy or alter evidence. Teeth and bone fragments and stomach contents are samples that can be safely stored in plastic containers before processing further.

With easily transported wet items, the item should be sent to the laboratory in a plastic or paper package to prevent contamination of other items and dried immediately on its arrival at the lab. The sample should then be repackaged in the original bag if possible or the air-dried original container should be repackaged with the air-dried evidence. Wet articles can be dried in the lab in drying cabinets before repackaging.

If a stained item is large, the stain can be absorbed onto cotton squares, transported to the lab and then immediately air dried before repackaging. Once in a secure location, wet evidence must always be allowed to completely air dry (unless protocols require the evidence to be stored in a freezer) before repackaging in a new, clean, unused, dry paper container. Biological fluid reference samples must be packaged in leak-proof containers.

Items that may cross contaminate each other must always be packaged separately, and containers should be closed and secured to prevent the mixture of evidence during transportation. To establish and maintain chain of custody, each container should have the collecting person's initials; the date and time the sample was collected; a complete description of the evidence and where it was found; and the investigating agency's name and file number

 Sexual Assault

In sexual assault cases the victim often provides information that helps in the identification, collection and preservation of the appropriate evidence. In many cases the victim may be hospitalized or taken to the hospital for the purpose of obtaining appropriate samples using a rape kit or sexual assault identification kit. These pre-constructed kits contain the swabs, documentation and materials necessary for obtaining and recording the appropriate samples. Typically, samples obtained from a victim will include vaginal, oral and rectal swabs, which are air dried before packaging. Swabs should always be obtained as soon as possible after the incident since critical evidence can be easily destroyed or lost through degradation and drainage from the body. Unfortunately, it often takes several days for a victim to report an incident, resulting in the loss of the most critical evidence. Quite often the victim will bathe repeatedly after an incident (often one of the first reactions of a victim) washing away the evidence.

Depending on where the suspect ejaculated, most useful case samples are obtained from the vaginal, cervical, rectal, and oral body cavities of the victim. The victim's underwear can also be a good source of evidence since, over time, seminal fluid will drain from the vagina or anus. Other items of evidence may include clothes, bedding, blankets, comforters and furniture and car upholstery, that can be a sources of seminal fluid or trace evidence, such as hairs or fibers, that may have been transferred from the suspect.

Searching large items like bedding and comforters for the presence of semen stains can be hampered by detailed patterns or the color of the item. Seminal stains can appear yellowish on a white background, but may be difficult to see on a highly patterned fabric. Side lighting or special light sources can be used to locate or highlight stains. Once a semen stain has dried it may leave a crusty stain. These stains can sometimes be located by just gently touching or running your fingers over the surface of the item (the crusty touch test). As before, all wet stains should be completely dried prior to packing. The investigator may outline wet stains with a wax pencil on larger items like sheets and bedding, which helps the lab analyst locate the stain once the item is back in the laboratory.

Preservation of Evidence

Storage

Blood and other biological evidence must never be exposed to excessive heat or humidity. Whenever possible, bloodstained evidence should be refrigerated until it can be transported to the crime lab. Evidence dried and sealed in brown paper can be safely stored at room temperature; liquid reference samples should be stored at 4°C. Cut out stains, awaiting further testing, are best stored frozen and are best packaged in heat sealed plastic, because regular paper will become damp in the freezer as it absorbs moisture. Before heat sealing, ensure all the air is pressed out of the package. This is another example of one of the few samples that can actually be stored or transported in a plastic container.

For further information on the collection and preservation of biological samples, go to the following website: Collection and Preservation of Blood Evidence from Crime Scenes at  http://www.crime-scene-investigator.net/blood.html

The following sections are particularly helpful:

  • Preliminary Considerations
  • Collection and Preservation of Blood Evidence
  • Dried Bloodstains

Also see Special Considerations for Sexual Assault Evidence at  http://www.crime-scene-investigator.net/evidenc4.html

Chain of Custody

Chain of custody is the record of evidence recovery, handling and analysis that is required by the courts for any evidence submitted in a trial. The following information must be recorded for chain of custody purposes:

  • a description of the evidence and its container
  • the specific recovery location of the evidence
  • case numbers
  • the date and time it was collected who collected it whether or not the evidence container was sealed upon transfer to another individual
  • who received the evidence
  • the dates and times of any evidence transfers
  • who delivered the evidence
  • the final disposition of the evidence.

This information is required to demonstrate that the evidence was not contaminated in any way that alters the information that the evidence originally contained. It also demonstrates that the original evidence was not planted or changed in some way before its presentation in court.

Sources and DNA Content of Biological Samples

Before analysis of evidence can begin, the biological samples must be collected from a known contributor such as the victim or perpetrator/suspect or as evidence from a crime scene. The table below lists some of the types of physical evidence that can be found at crimes scenes.

Type of Physical Evidence

Biological Source

Cigarette

saliva

Toothpick, dental floss

saliva

Stamps and Envelopes

saliva

Bottles, cans, cups, glasses

saliva

Condoms

semen, vaginal and rectal cells

Bedding

semen, sweat, hair, urine, blood, saliva

Bite marks

saliva

Fingernails

skin, blood, sweat

Tape and ligatures

skin, blood, saliva

Bullets/projectiles

blood, tissue, skin

Clothing

blood, sweat, semen

Hat, Mask,

sweat, hair, skin/dandruff

Knife, bat, other weapons

blood, skin, tissue

“Through and through” bullet

blood, tissue

Used condom

Semen, vaginal or rectal cells

Facial tissues, cotton swabs

Mucus, blood, sweat, semen, ear wax

Eyeglasses

Sweat, Skin

Source: Forensic analysis of biological Evidence: A lab guide for serological and DNA typing. J McClintock

Whole Blood

Whole blood, which is used as a reference sample for DNA analysis, may be obtained from the victim or when the suspect is in custody. Whole blood samples should be collected in a sterile tube that contains the preservative ethylenediamine tetra acetic acid (EDTA). EDTA also inhibits the activity of the enzymes known to degrade DNA. For short term storage these tubes can be refrigerated and frozen for long term storage. In some agencies the blood is spotted onto an FTA collection card which is an absorbent cellulose based paper saturated in chemicals which inhibit bacterial growth and prevent DNA degradation. FTA is an acronym for fast technology for analysis of nucleic acids. FTA papers are allowed to dry and can be stored at room temperature for years.

Bloodstains and Mixed Stains

When blood stained clothing is recovered then care must be taken to ensure the wet items are dry before packing to prevent contamination and cross transfer of staining which may deter the reconstruction of events.  In the short term such evidentiary items can be stored at room temperature in a humidity controlled environment away from direct light.  For longer term storage items must be dried and stored in a low temperature frost free (no humidity) freezer.

Hairs

Generally hair samples come in the form of head hair or pubic hair.  12-24 hairs collected from the scalp is usually sufficient for DNA analysis. Hair samples with intact roots provide sufficient DNA for STR (short tandem repeat) typing. A hair shaft contains enough mitochondrial DNA for mtDNA (mitochondrial DNA) typing. Hairs can be stored in the short term in a humidity controlled room at room temperature, or in a low temperature frost free freezer for longer term storage.

Swabs

Swabs may be obtained from wet biological material such as blood droplets, vaginal, rectal and buccal cavities. Inanimate objects may also be swabbed to recover biological samples such as cigarettes, envelopes, and bottles. Cotton swabs must be sterile and can be moistened to collect dried samples by swabbing the area of interest. Moist swabs must be dried prior to packaging or placed in a vial containing a small aliquot of sterile 1x TE buffer for short term storage. Swabs with biological material are stored in the same way as biological stains.

Bone, Teeth and Tissue

When circumstances result in low or insufficient in amounts of biological material such as blood and semen,  for analysis, due to degradation, damage, availability or inaccessibility, the analyst may turn to bone, teeth, skin and tissue to obtain sufficient sample.  Generally a section in the region of 1cm2 is enough for testing.  Once samples are collected they should be frozen and transported to the laboratory on ice and kept frozen until DNA analysis begins. Note that repeated freeze thawing can greatly compromise the sample.

Tissue Samples, Smears and Slides

When known biological samples are unavailable but needed as part of an investigation, it is also possible to use medical samples as a source of sample. In some cases analysts have been able to obtain useful profiles from stored biopsies or stored tissue from surgical procedures including pap smears and histological sections.  Usually one smear, slide or section of tissue is sufficient for successful DNA typing. These samples can be stored at room temperature indefinitely prior to DNA analysis.

Although it's labor intensive DNA can also be isolated from paraffin embedded tissues and the yield is often low and impure. Recently xylene has been used for paraffin removal which eliminated some extra handling steps that have been attributed to sample loss in past methods resulting in a slightly higher sample yield compared to earlier processes.

Semen and Sperm

Sperm collected from a vaginal swab will also contain epithelial (skin) cells from the female and possibly the male.  In these situations the sperm can be differentially separated from the skin cells using extraction methods.  When samples are thought to contain only spermatozoa, such as ejaculate recovered from a used condom, the sample can be collected and processed in the same way as described above for bloodstains or mixed stains. Sperm specimens should be stored frozen prior to analysis and multiple free thaw cycles should be avoided.

Urine

Urine is similar to other biological samples such as sweat and sebaceous oils, in that once it is concentrated it will contain a sufficient amount of epithelial cells to generate a DNA profile.  A minimum volume of 10ml will generally be sufficient, but when available as much as 30 ml would be optimum. Urine samples should be stored frozen and refrigerated for a short period of time prior to analysis, again avoiding multiple freeze-thaw cycles. Concentrated samples can also be collected from a stain or a swab from a known source. 

Fingernail Clippings

Fingernail clippings can be a great source of biological material for DNA analysis. Clippings collected from the victim or crime scene can be used to identify a suspect as well as predict the gender of the attacker. During a sexual assault the victim may scratch the attacker while defending themselves and collect skin and blood under their fingernails. Nail clippings and the underside of nails can be swabbed using a sterile, moistened cotton swab.  The swab is dried and placed in a container for storage or placed in a vial containing an aliquot of 1x TE buffer for short term storage prior to DNA analysis. Swabs can be stored long term in the way as stains.

DNA Content of Biological Samples

Sample/ Source

DNA Content ( approx)

Liquid blood

20-40 ug/ml

Blood stain

250-500 ng ( from 1 cm2 stain)

Semen

150,000-300, 00 ng /ml

Post Coital Vaginal Swab

0-3000 ng/ml

Saliva

1000-10,000 ng/ml

Oral Swab

1000-1,500 ng/ml

Hair (root)

1-750 ng/ plucked root

Hair (Shed)

1-12 ng/hair

Urine

1-20 ng/ml

Bone

3-10 ng/ml

Tissue

3-15 ug/mg

Fibroblasts

6.5ug/1x106 cells

Source: Forensic analysis of biological Evidence: A lab guide for serological and DNA typing. J McClintock.

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