Understanding Serology and Biological Evidence (5) | Fresno Criminal Defense Attorney

Understanding Serology and Biological Evidence (5) | Fresno Criminal Defense Attorney

Species Typing:  

One of the questions a Defense Attorney must ask is whether the stain was from a human.  Many times animal blood is mistakenly identified or believed to be human in origin.  Today, we have a scientific foundation to aid in the defense of individuals accused of a crime when blood is mistakenly believed to be from a human, but in reality was from an animal.  If blood was involved, or alleged to be involved in the facts or your case, having a Defense Attorney who is educated on the science involved in your case can help you present an intelligent, scientific defense to your case.

Prior to the advent of forensic DNA analysis, once the nature of a biological stain was identified, species testing was the next logical step as a means of determining whether the stain was human in origin. If the stain or sample was not human then the species of origin had to be determined. Some laboratories elected to miss this step and immediately conduct ABO blood grouping which itself is a test of species origin (only humans have the ABO typing system). In cases that involve wildlife and animals it is valuable to be able to identify the species of origin, and in the majority of cases nowadays, this is achieved using DNA analysis. The need for species identification was heavily utilized when processing the evidence from the mass disaster that involved the bombing of Pan AM 103 over the skies of Lockerbie, Scotland in 1988, when DNA analysis in Europe and the US were very much in its infancy. Brought down by a terrorist bomb, debris and wreckage from the flight landed in a rural area inhabited by humans and livestock. As part of the human identification process, each tissue fragment and stain recovered at that crime scene had to be logged, species typed, and then blood grouped if the sample was determined to be human in origin.

The principle of species typing depends on being able to identify blood proteins that are specific to each animal species. Most of the species tests conducted are immunological in nature relying on interactions between antibodies and antigens. An antibody is a protein produced by the body's B cells in response to the presence of a foreign body, or antigen, such as a bacterium or virus. Antibodies form the primary immune response, and function to prevent or minimize disease by attaching themselves to the foreign antigen. Then, through a cascade of biological actions, the antibody causes the antigen to weaken or be destroyed.

At the time, two of the most utilized techniques in forensic serology were immunoprecipitation and electrophoresis. These techniques were routinely used singularly or together in species identification. The antigen–antibody principle was used to produce a precipitin type reaction between the specific antigens present in an extract of a test stain and antibodies present in the reaction antiserum (a serum solution containing antibodies that are targeted against specific antigenic substances).  In immunoprecipitation reactions, an antibody combines with an antigenic determinant and a visible precipitate forms.


Specificity is the capability of an antibody to recognize only one particular antigen and is one of the most important parameters in species identification. Cross reactivity of an antibody can occur in two ways; the antiserum is not tissue specific and will react with different tissues from the same species, or, it's not species specific and will give reactions with other or closely related species ( such as sheep with goat). This cross reactivity between species occurs because relationships between species are reflected in similarities in protein sequences that have not changed significantly over the course of evolution. For that reason some proteins are better than others for identifying species specificity.

Ouchterlony Double Diffusion

The most popular species determination test is the Ouchterlony double diffusion test which works on the basic principles of immunodiffusion and subsequent precipitation. The Ouchterlony procedure involves adding antigen and antibody to several separate small wells in an agarose gel. The antigen and antibody radially diffuse from each well towards each other, forming precipitin lines where a specific antigen - antibody reaction occurs. The precipitin band formation provides information regarding the identity, partial identity, or non-identity of the antigen and antibody.

DNA Testing

Today, most forensic laboratories use DNA analysis to determine species origin. In this case, instead of identifying the presence of human protein they are identifying the presence of human DNA. Many labs are set up such that they no longer perform any immunological testing or they have a case screening section which performs the presumptive and species testing and a DNA section that conducts DNA profiling.

This technique of species identification uses a biotinylated oligonucleotide probe that hybridizes to human DNA that has been immobilized on a nylon membrane. The probe - specifically probe D17Z1 (aka SW49) is a 40-mer (40 base) oligonucleotide that is complementary to a primate specific alpha satellite DNA sequence on chromosome 17. Alpha satellite DNA is composed of highly repetitive sequence elements found near human chromosome centromeres, and is usually chromosome-specific.


More frequently encountered stains such as blood, saliva and semen will give similar results with both immunological testing and DNA testing. Overall DNA tests may be less prone to false positive results than the immunological tests, since the immunological methods are prone to cross reactions. Samples where the amount of available DNA falls below the limit of detection of the test should only be reported as inconclusive. If semen stains are aspermic then the DNA results will depend on the amount of other non-spermatozoa cells present and the size of the sample. In this case the DNA sample may give a negative result, but an immunological test may provide a positive result. An immunological sample that tests negative for human origin can be further tested with commercially available anti-animal antisera. DNA probes complimentary to non-human alphoid sequences are not normally used.

Application in Wildlife Crimes

As well as the application of these techniques in the determination of blood, biological tissues, or fluids of human origin, these techniques are also applicable in cases investigating the violation of wildlife laws, such incidents of poaching, dog fighting and Santeria religious rites. Routinely encountered samples in such cases include, blood samples, tissue sample and carcasses.


Electrophoresis is a separation technique that utilizes the fact that an electrically charged molecule placed in an electric field will move towards the electrode with the opposite charge. Positively charged molecules will migrate to the negatively charged cathode and negatively charged molecules will migrate towards the positively charged anode. A mixture of molecules with different net charges placed into the electric field will result in each charged molecule migrating towards the appropriately charged electrode. Because of the magnitude of each charge, the molecules will separate and migrate at different rates.

Gel electrophoresis was used in a forensic setting for determining the presence of biological markers such as polymorphic enzyme systems (more on that later) in suspect stains, as a means of personal identification and in the early processes of DNA sequencing DNA analysis for identification ( DNA is negatively charged). Today, DNA analysis is conducted using capillary electrophoresis and gel electrophoresis is still used in some research facilities for protein analysis.

The net charge on any protein depends on the amino acid composition of that protein. Amino acid substitutions on a protein will ultimately result in a change in charge of that protein. Electrophoresis can be used to detect any subtle variations in protein charge as a result of amino acid substitution; even those proteins that are closely related and differ only by one or two amino acids.

Once protein bands are separated by electrophoresis they need to be visualized. Some proteins like hemoglobin are naturally colored and can be visualized quite easily once separated in an electrophoretic gel. However, most proteins need to be enhanced visually through the use of specific staining methods. Proteins can be non-specifically stained using a number of readily available protein stains. Such stains include Ponceau red, Amido black, and Coomassie blue. Many specific staining methods also use antibodies or enzymes to target and identify specific proteins.

Blood Typing and Identification

In 1901 Karl Landsteiner made a significant discovery when he determined that all human blood was not the same but could be classified by specific type or group. This discovery resulted in a dramatic decrease in deaths resulting from blood transfusions, and paved the way for a new scientific discipline and significant advances in personal identification.  Blood is a complex mixture of cells, enzymes, proteins and inorganic substances.

Blood typing has gone forward and advanced to the stage it is used routinely in many criminal and sex related crimes.  As with a fact or science used to forward a criminal allegation, it too can be used to help a defendant mount a successful defense to criminal charges.  However, you would 

Red Cell Antigens

The first blood group system to be identified was the ABO system; discovered by Landsteiner through experiments in which many blood samples were separated into cells and serum. Suspensions of each cell sample were then added to each of the sera. In some instances the red cells clumped together, in a process now known as agglutination, brought about by a reaction between antigens present on the surface of RBC's and complementary antibody carried in serum. The red blood cell antigens are also called isoantigens - these are antigens found in certain individuals that are immunogenic in some other members of the same species.

The table below lists the antigens (Ag) and antibodies (Ab) that represent the four common ABO types:

Antigens (Ag) and Antibodies (Ab) representing the 4 common ABO Types

Red Cell Ag

Serum Ab

ABO Type







A and B




A and B


In the reproductive process, thousands of genes are passed onto each of us from our parents. The expression of these inherited genes determine everything from our gender, to the color of our eyes, to our blood type. The genetic description of an attribute such as eye color or blood type is called a genotype, the resulting product of that genotype such as the shape of a nose eye color, or blood group is called the phenotype.

Every one of our genes sit on a chromosome in a specific locus or position. A single allele (an alternative form of a genetic locus) for each locus (where a gene is located on a chromosome) is inherited from each parent. For example the allele at the locus for eye color may result in blue or brown eyes.

There are three different alleles that determine ABO blood group: A, B and O

Each of us has two blood type alleles, one inherited from each of our biological parents. Since there are 3 different alleles there are 6 possible genotypes for the ABO system:

There are 6 different genotypes: AA, AB, AO, BB, BO and OO; and four different phenotypes: A, B, AB and O.

Phenotypes and Genotypes of the ABO System






AA or AO


BB or BO



It is the phenotypic expression of these inherited genes that produce the individual structural differences between the ABO red cell antigens .The precursor to A and B antigens is the H antigen. Genetically controlled structural modifications of the H antigen produce a phenotype of group A or group B. The B gene produces a transferase that adds D-galactose to the structure of the H antigen, whereas, the A gene produces a transferase that adds N-acetyl-D-galactosamine  to the terminal D-galactose of the H antigen. The O gene doesn't produce any enzymatic activity, so the H antigen remains unchanged.

The frequency of occurrence of the four main ABO blood groups varies between, and throughout, populations throughout the world.

Frequency of Occurrence of the 4 main ABO Blood Groups

Blood Group

Phenotype Frequency UK

Phenotype Frequency US













Secretor Status

Some people also express their ABO antigens in a water-soluble form that can be found at high concentrations in body fluids such as saliva and semen, sweat, tears, and serum. Group O people will secrete H antigen; group A people will secrete A and H antigen, and group B people secrete B and H antigens. Soluble or secreted antigens are usually referred to as substances. The secretion process is genetically controlled and those who possess the secretor gene are called secretors. Approximately 80% of people are secretors and 20% are non-secretors. 

ABO Cell Grouping

Whole blood, or liquid blood samples, can be typed by separating the cells from the serum, and observing the agglutination of the separated cells by the addition of a known antiserum. Alternatively, samples of known cells can be tested for agglutination with the serum fraction. When testing whole blood samples, only two antisera are needed for a routine blood type determination: Anti A and Anti B antisera. Blood of group A will be agglutinated with the addition of anti-A serum; group B blood will be agglutinated with anti-B serum; AB blood will be agglutinated with both anti-A and Anti-B serum, and blood group O will not be agglutinated with either Anti-A nor Anti-B. This is called direct agglutination.

ABO Stain Grouping

Most bloodstains are encountered in the forensic lab in dried or stain form. Grouping of stains is more complex than the other samples already mentioned since red cells in blood stains are ruptured or lysed. Grouping of stains may also be made difficult because of stain or sample contamination; limited amounts of sample; exposure to harsh environments, and substrate interference like that encountered with strong detergents.

Blood Enzymes and Proteins

The antigens, polymorphic enzymes and protein subtypes that can be used to identify an individual are the product of genetically controlled traits. These traits are inherited from our biological parents and are passed on from generation to generation. Enzymes are proteins that have specific functional roles in the body; responsible for regulating many biochemical reactions. Before the introduction of DNA analysis, forensic serologists were interested in a number of biological enzymes that exist in one or more different forms. These polymorphic enzymes can be separated into protein components called isozymes. This discovery allowed forensic analysts to further narrow the possible source of a bloodstain within a given population. Although the use of biochemical markers in identification has been surpassed by DNA analysis, information relating to these grouping systems is still important especially in light of the reanalysis of pre-adjudicated and cold cases. These systems were the basis of the forensic evidence that led to many convictions which are now being overturned through programs such as the innocence project, leading to the exoneration of many people who were wrongly convicted of crimes relating to biological evidence.

Polymorphic enzymes are enzymes which have slightly different structural forms, usually due to having minor differences in their amino acid composition, but have the same or similar functional properties. Prior to the advent of DNA analysis, forensic serologists used polymorphic enzyme patterns to help determine the origin of biological stains and fluids. Red blood cells, serum and other body fluids contain numerous polymorphic enzymes that provide biochemical markers that can be determined using gel electrophoresis. Differences in these enzyme patterns could be combined with traditional ABO blood grouping to narrow down sample origin.

Since antigens, enzymes and proteins occur independently of one another, the probability of a bloodstain having a specific combination of markers is the product of their distribution within the population.

For example: A type A bloodstain may have originated from 42% of the population. If the stain also contains the PGM 1 enzyme subtype (found in 58% of the population) and the EAP BA subtype (found in 48% of the population) then the stain could have originated from 11.6 % of the population (.42 x .58 x .48 x 100 = 11.6). The more factors found in a stain then the smaller the frequency of occurrence in the population and the more significant or more discriminating the results become.

The frequency of occurrence of each biological marker can vary significantly between regional, national and international populations, as well as between races. The distribution of any given marker within a cohort of Caucasian, Black, and Asian population within the US may be significantly different from the distribution between the same racial groups in the UK .

The following table lists many commonly used biological markers and their subtypes.

Commonly Used Biological Markers and Their Subtypes

Grouping System or Blood Factor





Adenosine Deaminase



Adenylate Kinase



Carbonic Anhydrase II


Erythrocyte Acid Phosphatase



Esterase D



Glucose 6 Phosphate Dehydrogenase


Glyoxylase I



Group-specific Component






Peptidase A

Pep A




6-Phosphogluconate Dehydrogenase






Marker Systems

Haptoglobin (Hp)

Haptoglobin is an alpha-2 globulin glycoprotein that readily forms a stable and irreversible complex with hemoglobin, and controls hemoglobin excretion from the body. Unlike most genetic polymorphisms which are due to amino acid substitutions, the polymorphic nature of Haptoglobin is due to unequal gene crossover, resulting in production of allelic proteins with substantially different molecular weights. The most commonly seen haptoglobin phenotypes are Hp1 (1-1), Hp2 (2-2), and Hp2-1 (2-1). Hp is a valuable marker for forensic serologists since it is very stable in dried stains.

 Phosphoglucomutase (PGM)

PGM is a phosphotransferase enzyme that catalyzes the reversible conversion of glucose-1-phosphate to glucose-6-phosphate during carbohydrate metabolism. PGM is found in plant and animal tissues and in microorganisms. In humans, the enzyme is found in large quantities in blood and semen, and in smaller amounts in vaginal secretions and cervical mucus. When stored under appropriate conditions the enzyme remains active for prolonged periods.

Group Specific Component (Gc)

Gc protein is also known as vitamin D binding protein; a glycoprotein in the alpha-2 globulin fraction of serum proteins, produced by the liver. Its presence may be decreased in the event of hepatic disease and pregnancy.

Studies on on Gc polymorphisms indicated that serum stains produced more reliable results than dried bloodstains. Studies with aged serum stains demonstrated that the GC protein in this sample type was significantly more stable than in aged bloodstains.

Erythrocyte Acid Phosphatase (EAP)

There are two distinct types of acid phosphatases present in human tissues. Red cell or erythrocyte acid phosphatases have a low molecular weight (about 15 KD) and can be found in the cytoplasm of other tissues.

 Distribution of Genetic Markers in Tissue

As a Defense Attorney that handles many sex related offenses, there are specific facts that come into play when trying to identify or challenge the identify of the perpetrator. Fresno is comprised of a diverse population, with diverse genetic markers.   Many of the genetic factors found in blood can also be found in other body fluids, and those found in body fluids may also be found in blood.

The presence of genetic factors in semen became forensically important in cases of rape and sexual assault. Some protein markers may be present in sperm as well as seminal fluid. Those in seminal fluid were of particular interest because genetic typing could still be conducted after a male has been vasectomized. Semen deposited in the vagina is usually well diluted before collection, so only those markers that are normally present at high levels such as PGM 1 could be easily detected. PGM can also be found in vaginal secretions, but it degrades rapidly and could confuse the interpretation of results. Semen stains were relatively easy to analyze since the dilution effect was less prominent, and if the stain was fresh enough, even sperm markers could be detected. 

Saliva contains a number of polymorphic markers that are found almost exclusively in the saliva. Only a few are found in blood or other body fluids. Other tissues that have polymorphic genetic markers include hair, tooth pulp, and inner ear fluid; these are samples that are often well protected in cadavers and could play a role in cadaver identification.

I'm sure you can see from the above article how if live sperm where found in the alleged victim, something as obscure as whether a person, or what percentage of males have had a vasectomy could be key in putting forth a viable defense.

Sample Handling and Storage

Many defense experts have testified that mishandling or improper storage of samples have led to contamination or an incorrect identification of the sample.  Protein markers found in biological materials deteriorate over time. Deterioration begins at the time a sample is shed and progresses thereafter as the sample is collected, stored, and processed. To maintain sample integrity, biological evidence was analyzed as quickly as possible once it was received by the laboratory. Appropriate handling and storage conditions were important in prolonging the longevity of each sample. Wet samples were prone to degradation though the action of microbiological processes and endogenous enzymes. Drying of samples and storage under refrigerated or freezing conditions significantly slowed these degradation processes. Non-enzymatic processes such as hydrolysis and disulfide exchange reactions can also modify proteins, subsequently affecting electrophoresis patterns.

Degradative reactions that occur in dry samples include photo-oxidation, direct oxidations, and free radical reactions.

Appropriate handling and storage protocols included keeping liquid samples cold or frozen whenever possible. Frozen liquid samples provide active markers for months to years depending on the marker. For dried samples, freezing enhances marker stability.

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