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Determining Stains Evidence

Used Of Technology In Determining Stains Evidence

Define Evidence

According to Oxford Companion to Law, exhibit is a document or other thing shown to a witness and referred to by him in evidence or in an affidavit. Evidence is defined as all legal means exclusive of mere argument that tends to prove or disprove the truth of the subject under judicial examination. Under Section 60(3) of Evidence Act 1950, if oral evidence refers to the existence or condition of any material thing including a document, the court may, if it thinks fit, require the production of that material thing or the document for its inspection. The material thing mentioned could refer to any form of stains obtained from the scene of crime or body itself. If observe stains suspected to be forensic importance, investigator officer notes down the location, size, type of stain and intensity of the stains by seeing if the fluid penetrate through the fabrics or any other materials

Picture 1 : Oral Evidence

What Are Stains?

Stains are usually formed in a particular pattern from any forms of liquid and more specifically the fluid either diluted or saturated, either sticky or non-sticky, either high or low viscosity to certain surfaces or any other materials. Behaviour of the liquid which flows downhill following the effect of gravity causing them to be split into droplets in any directions. It can be a pool, smear, spot or splash. Pool is formed from the liquid or fluid poured through and accumulated. Smears are usually formed by contact of stained object or unstained object and it tends to be superficial with very uneven intensity whilst spots or splashes formed by drops of fluid being hit at surface either perpendicularly or obliquely. The shape and appearance of stains and smears can give useful information about various factors connected with crime e.g. able to indicate the direction in which fluid comes from depending on the angle of impact, velocity, surface texture and drop size of fluid (Pua 2009).

In the aspect of forensic importance, a few of the bodily fluids including blood, semen, vagina secretion, saliva, sweat and tears can be used by the investigators or pathologists to determine a suspect’s or victim’s identity through a science called serology (Lyle 2004). Other than the above mentioned, stains could be originated from the contact of any melted compound or trace evidences such as paint, oil, drug, ink, dye, melted metal elements in gunshot or explosive residues, trace of hydrocarbon etc.


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Picture 2 : Examples of Stains


Type Of Stains

Dried Vs Wet

There are many classifications of stains and one of them is dried and wet stains. Dried stain usually is superficial with a thin film of fluid on the surface and it involves those liquid that easily evaporate off from the surface while leaving trace behind that might not be easily seen or have been exist for a long period of time ago or never been washed off properly. It could be any of the trace evidences as mentioned earlier such as dye or paint. Whilst wet stain found usually is heavy and saturated where the fluid goes right through the garment or the fabric is coated in fluid. It is normally a recent stain that easily recognized by naked eye and involved large volume of liquid that not easily vaporized such as blood etc


Dried Stain

Wet stains could be easily lifted from the surface using dry and sterile swab and every single swabs are closed in a separate tube with proper label whilst dried stains must be treated with care using fairly wet and sterile swab in the same way. There is some different terminology for dried and wet stains e.g. semen is a type of wet stain whilst seminal stain is the respective dried stain (Pua 2009).

Wet Stain

Biology Vs Non- Biology

Another type of classification is biological and non-biological stains.

Biological stain is originated from the living organisms and it is usually fluid or secretion from the body of the organism itself such as blood, saliva, semen etc. These biological stains are frequently found at the scenes of crimes, particularly violent crimes, or on the body involved. It can serve not only to link a suspect to the scene but to identify an individual with a degree of certainty that matches or surpasses the use of fingerprints. This degree of individuality is also useful in cases of questioned paternity and ancestry.


Biological Stain
Non-biological Stain

Whilst non-biological stain refers to those chemical compounds like ink, drug, gunshot residues and food stain such as tomato sauce etc. that are not really have identification value but could refine the search of evidences with forensic importance and narrow down the possibility of the intention of crime or cause of death during the investigation. Distinguishing biological and non-biological stains for example blood versus tomato sauce or ink is important and can be done using one of the following blood presumptive tests that either causing color changes such as Kastle-Meyer (KM), TetraMethylBenzidine (TMB), Leucomalachite Green (LMG) or causing fluorescent reaction such as Luminol and Flurescein tests. Nevertheless, there have some elements of interference for each of them (Lyle 2004). To eliminate the interference, another test has to be done at the forensic lab for confirmation such as Teichmann test, Takayama test or spectrophotometric method in which particularly used for blood confirmation.

Kastle Meyer



Human Vs Non – Human

Human and non-human stains can be further determined after confirmation of biological stains, for instance, human blood is comparable to other animal blood. In this case, Precipitin test is used to ascertain that blood is of human origin based on the antigen-antibody complex formation which produces a clearly visible cloudy precipitate. It involves use of human antiserum (serum containing antibodies specific for human antigens) to detect presence of human antigens in 2 ways i.e. either conducted in capillary tube or by cross-over electrophoresis. If result is negative, test can be repeated using antiserum prepared for other animals as antiserum is commercially available for a number of animals such as dog, cat, deer, cow, and sheep.

Precipitin test is highly sensitive and effective for testing dried bloodstains. Other than blood, spermatozoa examination for seminal stains can be used to distinguish between human and non-human origin as there are some specific characteristics for different species in terms of features of head and tail, length and width of the head, total length of the spermatozoa as well as under different staining methods used like H&E and ABE (Pua 2009).


Human Stain

Non-Human Stain

Type Of Test Available

To determine whether a retrieved sample actually is blood or some other substance, the serologist conducts tests of two basic types including presumptive or screening and confirmatory. Presumptive tests typically are cheaper and faster. When they are positive, presumptive tests indicate likelihood that blood or other fluid is present but don’t establish that as fact. That’s why confirmatory testing then is needed and to be certain. When presumptive tests are negative, blood or other fluid not present, and the more expensive and time-consuming confirmatory tests can be avoided (Lyle 2004).


Blood isn’t the only bodily fluid that can lead investigators to a criminal. Semen, saliva and, in rare cases, vaginal fluid may hold the key to unlocking a crime. During sexual assaults, semen and saliva commonly are transferred to the victim, the victim’s clothing, or nearby surfaces. The first step in locating these bodily fluids is an examination of crime scene with either an ultraviolet or laser light source which causes these fluids to fluoresce or glow as well. These materials must be carefully collected and preserved, because as moist, biological materials, they are susceptible to putrefaction from bacterial growth.


The search for semen at the scene of sexual assaults includes the corpse or victim, underwear, condoms, bed sheets and mattresses, carpeting and flooring. Tests for semen are either presumptive or confirmatory. Presumptive testing is generally based on the fact that semen contains a very high level of the enzyme acid phosphatase (AP).

AP Test

Other presumptive tests search for the presence of two other components of semen viz. spermine and choline. Each of these tests is positive whenever crystals form after the sample is exposed to certain chemicals. Confirmatory testing relies on the presence of spermatozoa using microscopic examination or presence of prostate-specific antigen (PSA) which unaffected by vasectomy (Lyle 2004).


On the other hand, saliva is an important bodily fluid to the forensic pathologist and it can be recovered from bite marks etc. and reveal ABO antigens, blood type and sometimes yield enough DNA source for profiling analysis. Saliva begins the digestive breakdown of starch into its simple sugars viz. maltose and dextrins in the presence of its primary alpha-amylase. Screening tests such as Phadebas Forensic Press test and Pink paper test can be applied, but unfortunately there is no confirmatory test exists for saliva (Lyle 2004).

Saliva Paper Test

Amylase Test

Detecting vaginal fluid is difficult, but it may be important in non-ejaculatory rapes and penetrations with foreign objects. Swabs may be taken from a suspect’s penis or from any suspected foreign object. Testing depends upon the finding of glycogen-containing epithelial cells.

Periodic Acid-Sciff (PAS) is a reagent that stains glycogen a bright magenta color in the cytoplasm of epithelial cells that line the vagina. However, young girl and postmenopausal women rarely contain glycogen-rich vaginal epithelial cells (Lyle 2004).


As discussed earlier, screening tests such as Luminol and Fluorescein causing blood to fluoresce or glow in the dark under ultraviolet light, revealing blood that can’t be seen with naked eye. Perpetrators often attempt to scrub walls and floors clean, erroneously assuming that if blood can’t be seen, it can’t be found. Fortunately, that isn’t true. The area of distribution is also clearly defined. Spurts, spatters, drag marks, foot and hand prints jump into view for further projection analysis and crime scene reconstruction. Fluorescein better suited for exposing bloodstains because it doesn’t react with household bleach and is thicker fluid that sticks better to vertical surfaces compared to Luminol (Pua 2009).

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Polylight is one of the screening equipment that able to emit some colours of light at different fixed wavelength in the unit of nanometer (nm). In the application of forensic, polylight is used to detect biological evidences like semen, saliva and blood or trace evidences like hair and fingerprints that are invisible to the naked eye. Currently, Royal Malaysia Police possesses 3 types of polylight including PL10, PL500 and Crime-Lite (CL). Most of the body fluid mentioned above can be detected using CL 400 – 430 nm (violet) with yellow goggles (455 nm), CL 430 – 470 nm (Blue) with orange goggles (495 nm), PL 415 nm (Violet) with yellow goggles or PL 450 nm (Blue) with orange goggles (PDRM 2009).


Crime Lite


After all, the main objective of stain evidence is to narrowing down the focus to find whose stain is it belongs to e.g. by determining its ABO blood type using absorption-elution method to draw out the remaining antigens in the dried stains. By simply typing the blood at crime scene or body, investigators narrow their suspect list and completely exonerate some suspects by using population distribution information for the four ABO blood types. It can be further individualized the sample as red blood cells contain more proteins, enzymes and antigens with such catchy names as Duffy, Kell and Kidd and intracellular enzymes such as adenylate kinase, erythrocyte acid phosphatase, and the very useful phosphoglucomutase isoenzymes (PGM). The more factors inherited independently, the more powerful the identification of the sample is.


Approximately 80% – 85% of population is secretors, meaning they emit proteins of their ABO blood type in all bodily fluids including seminal fluid, saliva and tears. ABO types found can be used to eliminate a suspect in a rape, but they can’t accurately identify the individual who secreted them. Hence, DNA testing at 16 loci must be employed to make a conclusive match (Lyle 2004).

Jenis ABO


  1. Evidence Act 1950. Law of Malaysia: Incorporating all amendments up to 1 January 2006.
  2. Lyle, D.P. 2004. Working with Blood and Other Bodily Fluids: Serology. 1st Ed. Forensic for Dummies  pg. 213 – 225. Indiana: Wiley Publishing Inc.
  3. PDRM Forensic Laboratory. 2009. Use of Polylight and Crime-Lite during investigation. National University of Malaysia NX 3033 Physicochemical Evidences.
  4. Pua Hiang. 2009. Presumptive Tests and Identification of Stains. National University of Malaysia NX 3062 Body Fluid Investigation.
  5. Pua Hiang. 2009. Receiving, Recording & Examination of Exhibits. National University of Malaysia NX 3062 Body Fluid Investigation.


Last Reviewed : 8 September 2015
Writer : Lai Poh Soon
Accreditor : Dr. Siew Shueue Feng