The World of Forensic Laboratory Testing

Pathology involves the study of changes in the body caused by disease or injury. Forensic pathology involves the evaluation of pathology issues that arise in public forums such as criminal investigations and civil litigation. Most forensic pathologists are experts in each of two major branches of pathology. The first is anatomic, which deals with structural alterations of the human body. The second is clinical, which entails interpreting and overseeing laboratory testing on body fluids and tissues, including chemistry, hematology, microbiology, and others.

During an autopsy, the forensic pathologist first conducts a “gross examination.” This involves detailed documentation of physical characteristics such as height, weight, color of hair/eyes/skin, any physical markings (scars, tattoos, wounds, etc.), or any other physical anomalies. The autopsy includes dissection and measurement of the internal organs. From these tissues, samples may be taken for microscopic examination. These samples may include blood, fluid from the eye (vitreous humor), urine, bile from the gallbladder, stomach contents, and solid organs such as liver, brain, and lung. Additionally, tissue samples are collected for toxicology testing and possibly for other laboratory tests, such as DNA typing, cultures for infectious disease, and various chemistry tests.

The fluid from the eye (vitreous humor) is particularly useful for determining the cause of death as it can be tested for a number of different substances, including drugs, toxins, and electrolytes to name a few. This fluid is easy to collect and quite useful in that changes in concentration of substances that normally occur after death take place relatively slowly in vitreous humor. The results may aid in the diagnosis of conditions or diseases in certain deaths due to diabetic ketoacidosis, dehydration, renal failure, shaken-baby syndrome, asphyxiation, and others.

Forensic testing for the ingestion of poisons or drugs can be critical to a criminal investigation. While knowledge of toxic materials spans several centuries, the ability to test for these poisons systematically was not available until the early 20th century. Today, forensic toxicology routinely involves alcohol and drug testing.

Toxicology in postmortem cases
A forensic toxicologist might get involved in a postmortem investigation if drug intoxication wasn’t previously suspected, in drug-related homicides, or if either a suspect or the deceased might have been under the influence of drugs at the time of the fatal event. In some deaths, notably accidental and homicidal ones, drug testing may determine if impairment was a factor in the fatal incident.

Forensic toxicologists may not only perform comprehensive testing for drugs of abuse (including alcohol), but also therapeutic drugs. Alcohol testing is routinely performed in nearly all traumatic deaths, such as motor vehicle fatalities. Toxicology evaluations for therapeutic drugs may be important in confirming a death. One such example may involve measuring blood concentrations of anticonvulsant medicines in a person with seizures. If the deceased had been under-medicated or noncompliant with a treatment regiment, thus subject to a seizure episode, it relevance becomes an important contributing factor to the death.

Lab analysis of several different types of samples from decedents first requires separating the substance of interest from the body fluid or tissue sample before identifying substances with different tests. When the lab identifies tissue as positive for a particular substance, it must confirm results with a different and often more sensitive and specific technique. However, the presence of a particular substance doesn’t mean it caused the death. The lab must determine the concentration present in the sample, and the forensic pathologist must interpret these data, along with the remaining autopsy findings, to make that determination.

Other uses of toxicology
While forensic toxicology often deals with postmortem specimens, it may also involve the living and issues related to drug toxicity. Testing not only includes investigations of driving under the influence of alcohol or drugs, but also testing for illicit performance-enhancing drugs in athletes and drug testing in the workplace.

Illicit drug use continues to be a big medical and social problem in the U.S. Members of the military, public sector employees, health care employees, transportation employees, and a growing number of private sector employees are often required to be drug tested before starting a new job or maintaining that job. Drug testing may also be required when securing new insurance policies. Prison populations may also be tested for illegal drug use, most frequently screening for the presence of amphetamines, marijuana, cocaine, phencyclidine (PCP), and/or opiates (morphine, heroin). Urine is still the most common testing source, but drug testing can also be done on blood, hair, sweat, and saliva.

More recently, genetic testing has been added to the forensic pathologist’s toolkit. Molecular testing of DNA from cells in a particular biological sample can be analyzed to determine the unique genetic make-up of any one person. Each individual inherits a set of genes (two copies, one from each parent) that is unique and distinctive as a fingerprint. In clinical settings, genetic testing is used most often to detect chromosomal mutations that may be present in an existing disease state or used to determine a person’s predisposition for a particular disease. In forensic settings, DNA typing analyzes the genetic material from two or more sources and compares the genetic sequences to determine the likelihood that the two samples are from the same source or from a relative. This can be applied in “identity” and “parentage” testing and may be used in civil or criminal proceedings.

DNA typing can be used to ultimately identify an individual and can be done on a very small amount of sample. Often, a swab of cells from the inside of the cheek (buccal swab), a drop of blood, or a small amount of tissue can be enough to isolate a sample of DNA. Also, DNA is relatively stable and is not easily degraded by some less than ideal conditions such as heat, cold, or drying. It is ideal for identity and parentage testing because a person’s DNA does not change during their lifetime and is the same for all the cells in the body. Except for identical twins, DNA is different in everyone.

In contrast to medical genetic testing, forensic DNA typing does not reveal anything about a person’s health or medical history. The areas of the DNA sequence that are tested have no known ability to predict health status. This type of DNA sequencing is not the same as the in-depth, highly complex, full genomic sequencing often heard about in the news.

As with other types of forensic testing, DNA typing must follow strict protocols for proper sample collection, maintaining a chain of custody, and testing procedures. In the U.S., the Federal Bureau of Investigation’s (FBI) DNA Advisory Board has implemented standards for laboratories performing forensic identity testing and AABB has developed standards for parentage testing. Both standards focus on testing and quality assurance.

Identity testing
Identity testing compares DNA sequences from two separate sources to determine if they are identical. The DNA found at a crime scene or on a victim is tested and compared to that of the suspected individual. Such determinations can be used to match a suspect to a specific crime, exclude someone as a suspect, link a suspect to several crimes (serial), link crimes with no suspect in common, or exonerate someone who has been falsely accused.

DNA is also used to determine identities that can’t be distinguished in other ways, as in the case of catastrophe victims, fragmented remains, isolated body parts, and decomposed bodies. Labs identify individuals through analysis of DNA extracted from samples such as blood, saliva, tissue, hair, or bone.

DNA sequences consist of chemical units called nucleotides that vary in size and make up a chain-like structure. In humans, DNA sequences are over 99% similar in structure, yet the small percentage that is different makes each person unique. Labs look at these DNA sequences for matches between the presented evidence and suspects based on sequences of small segments of DNA at different locations on the person’s total genetic makeup (genome). Generally, a match at thirteen sites confers confidence of identity “beyond a reasonable doubt.” That’s because a match at 13 sites is a rare occurrence. Because only one-tenth of a single percent of DNA differs from one person to the next, all of the locations tested for forensic DNA typing vary widely among individuals. The possibility of two people with the same DNA profile (except for identical twins) is extremely remote.

In forensic testing, obtaining DNA profile information is valuable only if there are other profiles to compare it to in order to establish a match, to exclude a profile, and/or to make an identification. In 1990, the FBI began using a computerized software program called the Combined DNA Index System or CODIS. This program is used by law enforcement officials to compare newly determined DNA profiles with existing profiles contained within the database known as the National DNA Index System (NDIS).

In criminal cases, if a suspected individual is convicted of a crime, his or her genetic fingerprint (a select list of DNA polymorphisms) is put into CODIS. DNA evidence from unsolved crimes is also entered. Using this system, matches in DNA profiles have helped in the investigation of over 100,000 crimes as well as in helping to clear some who have been wrongfully accused of crimes.

The CODIS databank is comprised of several different types of indices that are used for identification purposes. DNA profiles from individuals convicted of crimes are entered into the Convicted Offender index of the CODIS data bank. Some states have laws that require that the DNA profiles of anyone arrested be added to the Arrestee index of the CODIS system. Another index includes DNA evidence (e.g., blood, semen) from crime scenes of unsolved crimes. Other indices within this system include profiles of missing persons and relatives of missing persons that may help in the identification of found persons or in the identification of remains.

The segments of DNA used to create a “fingerprint” are called “short tandem repeats” or STRs. These STRs do not represent genes but regions that occur on stretches of DNA that lie between genes. Genetic information related to genes and inheritance patterns (e.g., medical genetic testing for disease risk) is not entered into the CODIS system and the CODIS system cannot identify a person’s physical traits or genetic risk of disease based on STRs.

For more on the CODIS system, see the FBI online brochure.

Parentage testing
DNA is also used to establish paternity or family relationships if this information is relevant to a criminal investigation or civil litigation as in seeking child support.

In cases where biological family relationships are at issue, DNA tests can either include or exclude a presumed parent, sibling, or other familial relationship that may exist through a mathematical estimation called a “parentage” or “relationship index.” The probability of the relationship is estimated through a process that combines the likelihood of the genetic test results, the physical characteristics that are the expression of gene combinations (phenotype) of the parties involved (for example, eye or hair color), with the probability of other “non-genetic events,” which can include information such as the location of the alleged parent at the time of conception, etc. The resulting parentage index and probability of relationship are generally admissible as evidence in court.

Sexual Abuse Cases
Routine testing in alleged sexual assault cases can include DNA testing in addition to tests for pregnancy and sexually transmitted infections (STIs) like syphilis, hepatitis B and C, gonorrhea, chlamydia, and HIV. When done within a few hours of the incident, these tests provide information about the victim’s health prior to the alleged assault, not health status after the incident. Due to the lag time between the initial exposure and the technical ability to obtain a positive test result, some of these tests may be controversial. Testing may be repeated six weeks to six months after the incident to help determine if the incident resulted in pregnancy and/or infection.

If the victim doesn’t remember events around the time of a sexual assault, the victim may be tested for “date rape drugs,” including flunitrazepam (RohypnolTM) and gamma hydroxybutyrate (GHB). Other tests may include those for alcohol and drugs of abuse. Evidence of intoxication can be used to discredit the victim in court.

Child Abuse
In suspected child abuse cases, laboratory evidence can help determine if an underlying health problem may be the true reason for suspicious bleeding or bruising. If a child has numerous bruises yet no history of significant trauma, a panel of tests to exclude bleeding and clotting disorders like von Willebrand disease or another clotting factor deficiency (factor VIII and IX deficiencies) may be performed. Evidence of such a problem could exclude the possibility of child abuse.

When it is suspected that a child has been sexually abused, samples may be collected for testing that may include DNA tests and tests for sexually transmitted infections (as described in the previous section). Detecting the presence of an STI may aid in the determination of whether sexual abuse has taken place.

Other laboratory tests that may be useful when evaluating for tissue injuries, such as abdominal, internal organ injuries, include liver tests (alanine aminotranferase and aspartate aminiotransferase), pancreas tests (amylase and lipase), and tests to spot blood in urine. A positive stool for blood (guaic test, fecal occult blood test, FOBT) can point to rectal bleeding caused by abdominal or anal trauma.