Understanding Serology and Biological Evidence | Criminal Defense and DNA | Fresno, Ca (3)

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

Searching and Note Taking in the Laboratory


Once evidence is received and logged in at the laboratory the case is assigned to an analyst who then processes the evidence following several routine procedures. First the evidence undergoes gross examination and biological and trace materials that may be of significance to the case are located and recovered. During this process, the integrity of evidence seals are checked; the analyst begins to make notes and records observations in an organized fashion similar to the search of a crime scene.

The detection and identification process begins with a physical examination of each evidentiary item that includes the application of screening or preliminary tests to all suspect stains. This is followed by confirmatory identification and finally the sample is submitted for DNA analysis:

The following examination procedure can be applied to most forms of evidence encountered by the forensic biologist, including items of clothing, bedding, some kinds of weapons, fabrics, textiles and items recovered from scenes, and vehicles.

Searching and Note taking

The analyst selects an isolated area to examine the evidence.

Wearing a clean lab coat, the bench area is covered with a clean sheet of paper prior to opening any evidence bags (this will catch any debris that falls off the item being examined) - this paper is changed prior to the examination of each item.

Packages of evidence are checked against the evidence list in the case file; where necessary, evidentiary items are divided into those obtained from the suspect, those from the victim, and those from the scene. Evidence from different sources such as suspect, victim, and scene, should be examined in separate areas/rooms to avoid cross-contamination of trace materials (hairs, fibers, etc).

Once an item of evidence is selected for examination, the evidence bag is opened carefully in a manner that preserves the original seal and signature.

The contents are carefully removed onto the paper, and the bag is checked to recover any trace evidence that may be lost in the packaging.

While making notes at each step, the item is sketched and carefully searched; obvious debris and potential evidence is recovered, recorded and packaged; fibers and hairs are mounted on glass slides and other trace evidence is packaged and submitted to the appropriate section for analysis (pieces of glass or paint may be submitted to the appropriate section (chemistry, criminalistics) for analysis.

The appearance and location of any stains or damage on an item are noted in the records as well as the location of any trace evidence that is recovered from the item under examination. Pre-drawn templates can be used for recording information on commonly examined items (such as articles of clothing). Notes can reflect the characteristics of the item such as color, manufacturer, size, any tears/damage, stains, etc. which makes it easy to identify in court.

Presumptive tests are carried out on suspect stains. If stains present on the clothing are negative, remainder of item needs to be swabbed and tested as well since not all stains are visible to the naked eye.

Areas of stain that test positive with presumptive tests are selected for confirmatory tests and DNA analysis; Genetic differences between a suspect stain and a reference sample taken from the possible source allows that source to be positively eliminated (this source could be a victim or an assailant).

Once examination is complete, the item of evidence is wrapped is wrapped in the exam paper it is opened on so as to make sure that no trace evidence is inadvertently discarded.


Composition of Blood

Blood is a complex mixture of cells, enzymes, proteins and inorganic substances. The fluid portion of blood, the cell free fraction or plasma, is mainly water and accounts for about 55% of blood content. The solid materials, that are mainly cellular, are suspended in the plasma. There are three main types of blood cells that make up about 45% of the total volume of blood:

Red blood cells (RBC), or erythrocytes, are unique in that the mature circulating cells have no DNA. These cells function to provide oxygen to tissues by transporting oxygen molecules as part of a hemoglobin complex. The total volume of red blood cells is called the hematocrit.

White blood cells (WBC) or leukocytes are involved in the body's responses to infection. One group of lymphocytes are responsible for antibody production.

The third kind of blood cell is the platelet. These blood cells are involved in the blood clotting process: blood clots when of one of the dissolved proteins, fibrinogen is converted to a polymer called fibrin that traps platelets to form blood clots. The liquid that remains when blood is clotted is called serum.

There are several different serum proteins, but about half consist of albumin. Albumin plays a major role in preserving blood volume by regulating osmotic pressure. The remaining serum protein, which include molecules such as antibodies are classified as globulins. The main salts circulating in blood are sodium, potassium and chloride ions, and other organic substances such as glucose, hormones and vitamins.

Blood circulates throughout the body through the blood vessels that make up the circulatory system. The high pressure arterial system, that helps propel the blood from the heart to the major organs of the body, is composed of vessels with strong elastic walls. The venous system that returns the blood to the heart is much less elastic and under much less pressure. It is this pressure and the structure of the blood vessel that determines whether blood oozes or spurts from an injury site. Damage to a high-pressure artery or arteriole can result in the characteristic arterial spurt.

 Locating Stains

Blood stains typically have a reddish brown appearance. However, blood stains can take on many appearances on different items of evidence. Blood may look different on a red fabric vs. white fabric vs. black fabric. The stains may be old and degraded and may have a yellowish-greenish appearance. Some stains may have been exposed to the environment and be mixed with dirt. The stains may have been washed and appear lighter than a fresh blood stain. All of these scenarios are commonly seen in routine forensic casework.

Although blood does not fluoresce under an alternative light source like other body fluids do, there are tools available to aid the examiner in looking for stains. Illuminated magnifiers are large magnifiers with a light source attached. This can aid in looking at items such as guns or knives, where the blood may be in cracks and grooves and not readily seen with the naked eye. It is also helpful on black or dark clothing since the dark fabric may mask the stains. Infrared lighting can cause many fabrics to reflect a large amount of the light. However, bloodstains generally absorb most wavelengths of visible light as well as near infrared wavelengths of 700 to 900 nm. This results in the fabric appearing gray or white and the bloodstains dark in color which can be seen clearly in black and white infrared photographs. Other tools such as a simple flashlight or a stereoscope can also be used.

The first step in processing an item for the presence or absence of blood is to perform a visual exam. Any areas of interest should be marked (if possible). If it is necessary, illuminated magnifiers or other tools may be used to aid in locating the stains of interest.

Next, each individual stain should be tested using the laboratory's chosen presumptive test for blood (such as Kastle-Meyer, Leucomalachite Green etc).  Sample can be obtained by rubbing the suspect area with a dry swab. The same method can be used to locate blood hidden in cracks such as between the handle and blade of a knife. Although very little stain may transfer to the swab the tests are sensitive enough to locate the presence of possible blood. All results should be recorded on the item and in the notes. It may be beneficial to draw a diagram of the item or take a digital picture where the positive areas can be marked and used for later DNA characterization. This documentation is also very beneficial in court if you have to testify to your findings. Sometimes where the stain is on an item of evidence can play an important role in the case.

If all areas tested are negative OR the item you examine has no visible stains of interest, a general mapping / swabbing technique can be used. This is done by sectioning off the item (using a marker or Sharpie), taking a general swabbing or swipe from each section and testing each one. Each area of the item should be swiped individually ( for example, front of sweater, back of sweater, front of right sleeve, front of left sleeve etc) in order to be able to narrow down an area when positive testing occurs.  If there is a positive reaction to one of these swabbings/swipes, then the examiner has narrowed down where that stain is on the item.  From there, smaller sections can be made to isolate the exact stain. If you are concerned about exhausting the stain by performing more testing, the general area can be removed and sent on for further characterization.

Once a possible blood stain has been identified, a portion of the stain (or all, depending on the size) can be removed from the original item for further characterization.

Common Collection Techniques

The type of collection technique you should use to remove a small portion of a stain for further testing will depend on the item that the stain is on. Some common collection techniques are listed below with examples of the types of evidence they can be used on.

  • Cuttings - Fabric (ex. clothing) or other soft, porous surfaces that can be cut (ex. paper).
  • Swabbings - Hard surfaces (ex. guns, knives) or fabric (especially fabric where the stain is easily swabbed like nylon).
  • Scrapings -Fingernails (scrape the underside surface for possible foreign debris or blood from an attacker or victim), and hard surfaces (if the blood stain is heavy it can scrape offin flakes).
·         Chemical Tests
·         Presumptive Tests
  • Presumptive tests are sensitive but not specific tests for body fluids that depend on the detection of constituent chemicals. Presumptive blood tests are rapid, chemical tests that may be used to locate and differentiate between blood and other similarly colored stains. Presumptive tests are highly sensitive, easy to perform and allow minute traces of blood to be located when they might not be easily noticed. They are sensitive enough to apply to areas of scenes that may have been cleaned in an attempt to hide the evidence.
  • Most presumptive tests for blood rely upon reactions associated with the peroxidase activity of hemoglobin. The most common tests depend on the oxidation of colorless reduced chemical indicators, many of which are known or suspected carcinogens. Most presumptive tests are color tests where the reaction with blood results in a color change in the reagent. Unlike the other color tests, the stain visualizing enhancer Luminol causes stains to chemiluminesce

Common Color Tests for Blood






Blue color



Tetra methyl benzidine

Blue color


Probable carcinogen


Blue color


Probable carcinogen

Phenolphthalein or Kastle Meyer test

Pink color


Relatively Safe

Leucomalachite green

Green color


Relatively Safe

Luminol (5-amino-2,3-dihydro-1,4 phthalazinedione)



Probable carcinogen

  • These presumptive tests are not specific for blood, since other substances possess peroxidase activity. These include chemical oxidants such as rust and vegetable peroxidases such as horseradish.
  • The usual test sequence is to add a drop of reagent to stain extract (a dry rub using a swab is fine); add a drop of hydrogen peroxide and the color change is observed. The rapid (< 20 seconds) development of an intense color is considered a positive result. Color changes that occur before the addition of peroxide or changes that take longer than 20 to 30 seconds to develop should be considered negative.
  • The most commonly used presumptive blood tests for blood include the Kastle-Meyer test (Phenollpthalein), and LMG (Leucomalachite Green). More recently Hemastix is also being used in the laboratory and in the field at crime scenes due to its ease of use.

·         KASTLE-MEYER

  • In presumptive catalytic blood tests such as Kastle-Meyer, the oxidant (hydrogen peroxide) oxidizes a colorless reagent to a colored reagent.  Heme catalyzes this oxidation by cleaving an oxygen from hydrogen peroxide (H2 O 2 ). The Phenolpthalein reagent is time consuming to make, because it involves a reflux reaction to turn the pink colored phenolphthalein to the colorless form phenolpthalin. This reagent can be made in the lab or can be purchased in ready-made kits.  Many crime scene investigators use pre-made kits for use at crime scenes.  Leucomalachite Green is a very similar test to Kastle- Meyer, it works on the same principle, but the reagent is easier to make in the lab.
  • The use of Hemastix as a presumptive indicator of blood is being used more frequently because of its ease of use.  This is a product originally developed to detect trace amounts of blood in urine samples in the form of prepackaged test strips. To conduct a test simply add a drop of water to the strip and then rub it against the stain in question.  The test strips are composed of diisopropylbenzene  dihydroperoxide and  3,3',5,5'-tetramethylbenzidine  (TMB) which is also a reagent used independently much like the Kastle-Meyer test.  The benefit to the strips is that they are a one step process and the test can be done fairly quickly without several reagents available (such as ethanol and hydrogen peroxide). The downfall to Hemastix is that the specificity is lower than other tests available and it is sometimes hard to get stain transfer to the tip of the strip (especially with diluted samples).
·         Commercial Kits

Many presumptive tests are commercially available in kit form for rapid, easy and low risk field-testing. Most field test kits for blood test contain the testing agent Luminol and one or two other presumptive test reagents in disposable chemical applicators. Kits also include test papers, sterile water, instructions and a Confirmatory Tests

Microchemical Tests

To confirm the identity of a positive presumptive test for blood, there are two microchemical tests that can be used to confirm the presence of blood through the production of characteristic crystals. The most common test is the Takayama or hemochromogen test. In this test, ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin.  Once the reaction occurs, the crystal product can be observed using a light microscope. The other confirmatory test is the Teichman reaction that works through the same principles as the Takayama test. Although both tests give comparable results, the Takayama test tends to be preferred, probably because it's easier to use and the same characteristic crystals tend to form with a range of stain types. These tests tend to be less sensitive than color tests and can be disrupted by dirt and other particulate matter present in the sample.


Sometimes laboratories will use microscopy to verify the presence of blood cells in a suspected blood sample. While this is a straightforward process when dealing with whole blood, it's much more difficult when dealing with dried bloodstains. With dried stains, the cells must be reconstituted, re-dried, fixed and stained for microscopy for the identification of characteristic morphological features. Successful identification using this technique really depends on the level of expertise of the crime lab analyst and their ability to find intact cells within the stain. The integrity of dried red blood cells can be affected by the age of the stain, the condition of a stain and the nature of the surface the stain is on.


Other tests for blood rely on the use of spectroscopy to distinguish between hemoglobin and hemoglobin derivatives such as methemoglobin, carboxyhemoglobin, sulfhemogobin and oxyhemoglobin. This is considered to be a reliable technique that has been shown to give results with older stains that have tested negative using color or microcrystal tests. All hemoglobin derivatives exhibit a strong absorbance at 400-425 nm (called the Soret Band).


The most commonly used confirmatory test for the presence of blood is the ABAcard Hematrace test.  This is a test that detects the presence of human hemoglobin in a sample.  It is based on an antibody-antigen reaction between the sample (hemoglobin = antigen) and the antibodies in the test card. This test works much like a pregnancy test and looks very similar. Not all labs use this test because the Hematrace card has been shown to react positively with ferret blood. For this reason some laboratories will not use it as a confirmatory test for human blood. Other laboratories still use this test to confirm human blood but may put a disclaimer in the lab report indicating it's cross reactivity with ferret blood.

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