How to Become a CSI
The Fluorescein Method of Latent Blood Detection
Ricardo Tomboc, CLPE, CSCSA
Abstract: I was not highly impressed when the Fluorescein Method was first introduced in 1999. I was skeptical, resistant to change, and you can pretty much say that I was set in my ways. Much like my first impressions of Digital Photography fifteen years ago. Who needs change! I've read articles on Fluorescein by Robert Cheeseman and even spoke to him on several occasions. And the more I understood the process, the more I was willing to try it. Then I had a homicide case that required the need to develop possible latent blood from the rear seats of a suspect vehicle. We did not have any Luminol in stock, so I called a colleague, Randy Beasley, Forensic Specialist II from the San Bernardino Sheriff's Office (now retired), who gave me a pint of Fluorescein and Hydrogen Peroxide in small handheld pump spray bottles along with some quick verbal instructions. I was impressed! In just a few minutes I was able to set-up, apply the Fluorescein, and record my findings. Although the results were not what we were expecting, several smaller suspected stains were located.
Trying to find traces of latent blood at a possible crime scene is always very challenging, especially when the supposed crime scene has been thoroughly cleansed several times. There are several options available to the Crime Scene Forensic. BlueStar, Luminol, and Florescence are only some of the more particle techniques for use on larger scenes. Although I will be focusing on the Fluorescein Method of Latent Blood Detection; the other techniques may be just as viable.
Fluorescein can be obtained in various forms. Depending upon the formulations that are being followed, mixing a batch can be complex. However, there are prepackaged kits that can be obtained from various vendors that make it as simple as "just adding water"! Two such kits are sold under the registered trademark HemaScein, and another vendor under the trademark Flora-Scene. Since HemaScein, Flora-Scene, and Fluorescein are the same basic formulations, this paper will refer only the chemical name Fluorescein.
Fluorescein is a presumptive blood test for latent bloodstain detection. It has been used in forensic applications to reveal trace amounts of blood. Traces of latent blood can be detected even after repeated cleansing of the crime scene. Fluorescein is highly sensitive to the hemassociated molecules (enzymes and iron) in the red blood cells, (1:105,000 depending upon the dilution rate of the working solution) [1-2]. Traces of these hemassociated molecules will embed themselves on the substright, even after multiple cleanings. Fluorescein can also be used to discover and enhance shoe tracks leading from a bloody crime scene thus allowing investigators to follow the suspect's trail . Fluorescein can be used to locate traces of latent blood on clothing, even after it has been laundered several times (best preformed at the lab). It can also be used on vehicles (inside and out) . Some studies have been conducted on the development of bloody latent finger/palm prints . There are two basic forms of Fluorescein that are typically used in forensics.
Fluorescein can be either aqueous or alcohol-based. Both formulations will work. However, alcohol-based solutions are more difficult to make, and can be very volatile, and extreme caution must be taken when used. An open flame or static electricity can ignite the alcohol fumes possibly causing an explosion.
Both Fluorescein and Luminol are on the suspected list of carcinogens yet; Fluorescein has a Food and Drug Administration (FDA) approval for medical usage . Since the 1960's Fluorescein has been used to diagnose blood flow status in the retinas by injecting it directly into the vessels of the eyes, and as a topical used in diagnosis of corneal abrasions, ulcers, infections & dry eye [7-8]. Other medical applications of Fluorescein include injecting it into the blood system to study the blood flow to tumors, grafts, etc. (also known as Fluorescein mapping) along with other medical diagnostic procedures  [10-11]. Nevertheless, caution should always be employed when using either aqueous or alcohol protocols. The other chemicals used in the aqueous-based Fluorescein formula are zinc and sodium hydroxide, both of which can be commonly found in most households. Again, proper handling and disposal of all reagents should be employed at all times.
Some non-forensic, non-medical uses of Fluorescein have been "use as a water-soluble dye added to rainwater in environmental testing simulations to aid in the locating and analyzing any water leaks, and in Australia and New Zealand as a methylated spirit dye." Fluorescein has been used to dye the Chicago Rivers green on St. Patrick's Day .
Fluorescein reacts to the proteins and iron ions found in the hemoglobin and will fluoresce when exposed/excited to light at the 420nm to 485nm wavelengths peaking at about 460 to 480 nm range (blue light, near Ultraviolet).
The Fluorescein method of latent blood detection will require the use of either a Forensic Light Source (FLS), which can be Light-Emitting Diodes (LED); Alternate Light Source (ALS); strong Ultraviolet (UV) Light; barrier filter (deep yellow or orange); and a 1.5 to 3% solution of Hydrogen Peroxide (off the shelf concentration) used as a to catalyst to increase reaction times.
Application of Fluorescein can be accomplished by the use of either a simple spray bottle, garden pump sprayer with a fine mist sprayer tip, spray gun, etc. Airbrush sprayers can be used on smaller surfaces. The type of spraying equipment being used is not a major factor but, then again, the finer the mist one can obtain, the higher the visible quality and the greater the detail of the developed latent blood. For example, when a latent bloody shoe impression (or any other impression) is developed, the finer the Fluorescein mist, the finer detail will be .
With both Luminol and Fluorescein, the process must be accomplished in a darkened room or at nighttime. Ambient light must not be so strong as to overpower the fluorescent reaction during the analysis and photography. Some ambient light is good during the photographic process in that it can help to reveal the relevant areas surrounding the fluorescing stains, placing the fluorescent stain in the crime scene. If insufficient ambient light exist, a low-level flash can be used set at about 2 to 3 stops below the exposure used for the fluorescence alone.
Although there are many advantages in using the Fluorescein process; there are a few caveats that investigators should be aware of. These same factors can also be associated when working with Luminol and/or any other presumptive test. They are, but not limited to; Fluorescein must be used in a darkened area together with an ALS, or FLS at 420nm to 485nm, or a strong UV source (Luminol or Blue Star does not require an external light source). Another caveat is the possible hazardous-waste situation that applying and/or spraying any chemical substance can create. Obtaining the cleaning services of a hazardous-waste cleaning company should be considered to avoid any possible liability. The duration of the fluorescing reaction is only five to seven minutes, and may not be repeatable to any degree clarity. However, some experimentation has shown that once the treated areas are dry, a reapplication of the Fluorescein and hydrogen peroxide may be applied, but a shorter reaction has been noted (obtained from several direct informal conversations with Rob Cheeseman of RC Forensic).
The latent blood development process can be time-consuming, therefore one must ask, "What useful information can be derived from the application?" A positive reaction does not mean that the Fluorescein is reacting to blood. Both Fluorescein and Luminol can react to bleach, cleansers, cleaning materials, human and animal urine (which may not contain blood), certain types of metals (iron and copper), and/or any strong oxidizers . This type of reaction is known as a false positive. Another caveat is that the reaction time is limited and documentation must be done quickly. Repeating the process or re-applying the Fluorescein and/or Hydrogen Peroxide may weaken the reaction and may dilute any possible specimens. If processing on a metal surface, one must determine if the positive reaction is being created by latent blood or by the metal and/or rust that might be present.
Inherent fluorescence is a reaction to a substances that will fluoresce naturally when luminated with an ALS, before the application of the Fluorescein. Therefore, to avoid false positives due to inherent fluorescence, the scene should be analyzed for inherent fluorescence prior to processing the scene with the Fluorescein. Any areas found to fluoresce should be noted and/or marked to avoid any possible false positives after the Fluorescein is applied.
The fluorescence generated from the reacting Fluorescein and an ALS is called the target fluorescence. Inherent fluorescence is the fluorescence found at a scene that was generated from an ALS, and did not require any specific processing. Inherent fluorescence is most commonly from animal and human urine, semen, fluorescent paints, or any substance that will fluoresce under an ALS or ultraviolet light. Although inherent fluorescence and target fluorescence will both fluoresce, they may not fluoresce with the same intensity, and/or display a pattern that can aid in its identification. An experienced technician should be able to differentiate the two reactions.
Under an ALS, the oxidizing Fluorescein reacting to the substances on the substright will fluoresce. After approximately five minutes, the none reactive Fluorescein to fluoresce as well thus, decreasing the contrast between the target fluorescence and the background fluorescence. Background fluorescence occurs when the Fluorescein on the substrate oxidizes from the exposure to the oxygen in the air, and eventually the background fluorescence will blend in with the target fluoresce and loose any visually detectable trace between the two reactions. As mentioned before, reapplying more Fluorescein and/or Hydrogen Peroxide at this point may not significantly increase the target fluorescence and may tend to dilute any of the biological samples present (obtained from several direct informal conversations with Rob Cheeseman of RC Forensic). Caution should be taken because the over usage of the Hydrogen Peroxide may damage any possible DNA present. It is suggested that if a reapplication of the Fluorescein is required, apply only the Fluorescein and hold back on the Hydrogen Peroxide. Remember that Hydrogen Peroxide is used to clean-up blood and disinfect from wounds. It had been noted that after a thorough drying of the suspected area, a reapplication of the Fluorescein and Hydrogen Peroxide can be tried, however the reaction times may be shortened.
The literature has shown that Fluorescein will not affect DNA analysis . However, if a thickener is being employed, it is best to indicate that on any DNA samples collected. Thickener will not interfere with the integrity of the DNA. But, it can cause complications with the automated DNA extraction process. Thickeners are used to help the Fluorescein adhere to a vertical surface (rarely if ever used now). Recent studies have shown that a thickener is not needed if the application of the Fluorescein can be accomplished with a fine-mist sprayer.
Fluorescein is not the best method for locating whole visible blood. The best way of locating whole blood is with the use of a high intensity, high quality white light, and/or an ALS. There can be occasions where the search, and/or discovery of whole blood can be enhanced by the use of Fluorescein. For example, a large outdoor scene where small droplets of blood sprayed over a large area might be too difficult to see due to the color of the blood and the background/substrate. Keep in mind that the Fluorescein solution can dilute any present blood samples.
Whenever applying a latent blood detection test, one should always conduct a positive control test of the working solutions, and document the results. A positive control test is simply testing of a known a blood sample with the Fluorescein and Hydrogen Peroxide solutions, with the ALS, and a barrier filter. Each new batch of Fluorescein should be tested. It is recommended that a positive control test be done to any solutions sitting on a shelf for any length of time, before it is applied to any case work. This will reassure that the solutions are properly working. This also allows the investigators to differentiate between "The absence of evidence and the evidence of absence" . Is the target area truly indicating a negative result, is the testing procedure up and running properly? A positive control test can answer the question.
At older crime scenes, the ability to detect latent blood traces is paramount to the investigation, and may be critical to a successful investigation. At a newer crime scene where visible blood has been cleaned-up, again the results are crucial. The ability to interpret the results is as important as the results themselves. A positive reaction does necessarily mean that the Fluorescein is reacting to blood. As mentioned before, Fluorescein will react to bleach, powder cleansers, and other cleaning materials, human and animal urine, certain types of metals, and/or any strong oxidizers. Fluorescein, after a period of time, will eventually oxidize reacting to the oxygen air, even if applied onto a sterile substrate.
When investigating a recent crime, and the crime scene shows no obvious attempts of being cleaned-up (e.g. dust, dirt, pet hairs, etc. are layered on top the substrate, which might have taken several weeks, months, or years to accumulate); the Fluorescein process may not be appropriate for use by the investigator, since the process may not yield any results useful to the investigation. If reactions are detected, they may not be related, or part of the current crime. For example, small amounts of blood is expected to be found in bathrooms and in kitchens, but blood found in kitchens may not always be human blood; or positives reactions may be found on carpeted areas from animal or human urine.
Some of the best applications for Fluorescein are as follows:
To use the Fluorescein process or any latent blood-processing technique, when it is not necessary is costly, unfruitful, time consuming, and can cause many people to be inconvenienced. Misapplication of the process can also generate more questions than it can answer. Victims or suspects may not be receptive of having their homes exposed to any type of chemical processing. The exposure to potential liability is always present.
Although it is important to document the results we obtain with Fluorescein, the photographic process can be challenging to some. I don't place a significantly high effort into documenting the "fluorescing Fluorescein glow." Depending upon the case, I may choose to use only video to record my findings. Sometimes, I may choose not to use any video or photography, depending upon the circumstances of the case. I use the Fluorescein just as I would any other presumptive blood test to locate any possible latent blood (DNA).
When we use Hemident, Hemastixs, Phenolphthalein, or Leucocrystal Violet swabs, or any other presumptive blood test, do we always photographs our findings; taking close-up photographs of the color reactions on the swabs? Fluorescein is no different than any presumptive on the market. The difference for me is that I use Fluorescein to locate latent blood traces in large areas such as entire rooms, buildings, or vehicles. I would use the other swab type presumptive for smaller items, stains, or small spots.
However, there are times when photographing the fluorescing stains running down a wall or the swirling scrubbing cloth marks on a floor are very important to the investigation. In this instance, photography and video recording the process if very important!
As in all crime scene applications, every step of the process should be documented by some form. Photographic digital, and/or video documentation can be challenging. In a darkened room one must have the capability of using their still camera on time-exposure mode on a tripod. Videotaping using a camcorder with the ability to record at low-lux levels can be an excellent addition to or an option to still photography. Using a digital camera in video mode is also an acceptable alternative. However, making prints from a video recording will produce low quality prints. Again, depending upon the end use of the Fluorescein results; photographic, digital, and/or video documentation may not be required. If the Fluorescein process is used only to detect shoe tracks, videotape and/or a simple notation on your reports may be all you are required, depending upon your Agency's Policy and Procedures. However, good results have been obtained using Fluorescein to develop bloody latent shoe impressions.
Some helpful photographic hits for florescence photography is to have the camera on a tripod. Use a high ISO 800 plus if fine detail information is not being photographed as in a shoe impression. Expose for the fluorescing image and not for ambient light. Ambient light should be very low and not stronger than the fluorescence. Use a wide-angle lens since it will facilitate a greater depth-of-field. Use a aperture that allow sufficient depth-of-field, and a minimal shutter speed. There are so many factors to consider and each scene can be very different from the next that recommending a fixed shutter speed and aperture is not particle. That is why experience is so important. You get experience from practicing your skills before you get to the crime scene. However, on a typical Fluorescein practice session with a low-level ALS, in a very darken room, the following settings may be a good stating place: ISO 800, F5.6 Aperture Priority mode (Av or A setting) at 5 seconds. The flash can be set on TTL with the exposure compensation set at -3 or no flash required if sufficient ambient light exist. Don't forget to use a yellow or orange barrier filter on your lens.
The main objective of any latent blood detection process is to search for and ultimately obtain usable DNA. Secondary benefits are the ability to document that glowing fluorescing bloodstain patter showing where the DNA was obtained. However, without DNA, a glowing stain would be much less useful in court.
There are various ways of collecting samples after processing. When a suspected area fluoresces, that area should be noted by drawing a border around it with a marker, chalk, or other means. That area if possible should be cut out and preserved. A sufficient amount of sample should be collected to include a control sample. Remember that the collected samples should be air dried before packaging. If the area can not be cut out, than swabbing the area is an option. Remember that you are not collecting whole sample, but trace samples. Take the swab samples while the area is still moist. Scrub the area well with the swabs. As with all swab samples, don't forget to collect control samples from a near by area of the same substright; just as if a blood sample were being taken.
Investigators should adhere to all policies, procedures, and protocols. If no written policy or procedures are available to the Department, then the standard protocol listed on the instructional materials can be followed.
Below is a sample basic protocol outline:
*TECHNICAL NOTE: Fluorescein is converted into Fluorescin when mixing the stock solution. When the Fluorescin reacts with the proteins or iron ions, it is converted back of Fluorescein. In this paper, both Fluorescein and Fluorescin are being used synonymously.
Mixing of the Fluorescein chemistry is made simple when using the Flora-Scene™ Reagent Kits by RC Forensics (email@example.com) or HemaScein™ from Abacus Diagnostics. The process takes about five minutes or less when the thickener solution is not required. Thickener takes about one to two hours to mix, but it's not always needed. The Thickener was designed to keep the Fluorescein from running down vertical substrates. However, applying both the Fluorescein and Hydrogen Peroxide in a fine mist will accomplish the same task. Thickener is not offered in the HemaScein™ kits.
The HemaScein™ and Flora-Scene™ kits come with a vial of concentrated powdered Fluorescein and zinc, which has an expiration of seven years after purchase. To prepare a stock solution, a measured amount of distill water is added to the powered Fluorescein, and zinc and mixed vigorously together. This stock solution can have shelf life six moths to several years if the Fluorescein and zinc are kept in the same vial. To mix a batch of working solution, 1 ml of the stock solution is mixed to 100 ml of distilled water. If proper storage instructions are followed, the working solution can last as long as seven months. The keys factors to keep in mind to maintain a longer shelf life to the chemistry is to store it in refrigeration and protected from light, thus wrapping the solution bottles in aluminum foil. The stock and working solutions of Fluorescein are susceptible to oxidation, so limiting exposure to air and keeping lids airtight are important.
Sequential blood enhancement protocols can be employed. Amido Black, Leuco Crystal Violet (LCV), Coomassie Blue, Acid Yellow, and Ashley's Reagent, are just a few of the reagents that can be used after the Fluorescein process. In some instances, these other reagents may be a better first choice over Fluorescein. Some studies have indicated that the sensitivity of fluorescein to be better than the other dye stains or bloodstain enhancement techniques .
A few words on court testimony. Don't make the Fluorescein Technique any more technical than it needs to be. Fluorescein method is simply a presumptive blood test. It's testing for the possible presents of latent blood. Presumptive testing is done on blood all the time. The Fluorescein method can be validated if the collected samples come back positive to DNA.
If the suspected DNA collected, which was located from using Fluorescein, and does not come back positive for whatever reason, then it might be a little more difficult to determine that a suspected fluorescing blood patter is in fact blood. However, a Blood Pattern Expert, who has the expertise to intrepid the pattern as blood splatter, might validate the pattern. If the fluorescing Fluorescein stain has to stand alone in court, it might not have as much weight when presented, but at the very least it can be a piece of the puzzle that just has not been connected to the entire picture yet.
So if the value of the fluorescing Fluorescein presumptive, when validated via DNA, is maybe worth "ten nails in the suspects coffin, then value of the value of the none validated fluorescing Fluorescein might be worth one" in court, that is if the Defense us not able to have the fluorescing Fluorescein presumptive excluded.
I've recently had a court case where the age of the detected latent blood was in question. The crime scene was and outdoors scene and exposed to a desert type environment. I was asked by the defense, "can Fluorescein react to latent blood that is as old as two years"? The obvious answer was "yes." Fluorescein is expected to detect traces of latent blood that are several years old, however definitive testing has not been accomplished to determine exactly how old.
The Prosecutor jumped into the questioning and asked if environmental factors such as weather could have washed away all the traces of the latent blood during that two-year period of time? I answered the question that "in theory, I would expect to get some reaction from the Fluorescein; but several factors can determine the degree of the reaction, if any." Those factors would be the amount of rain; the original dilution of the blood deposited before it was washed away, etc.
The Defense attorney was concerned that a vehicle in driveway at the scene was not processed with the Fluorescein. Defense asked if blood washed off a car could still be on the driveway at the scene. I answered the question the same way as before that "in theory, I would expect to get some reaction from the Fluorescein; but several factors can determine the degree of the reaction, if any." It would be up to the investigators to determine if processing a specific area is of value; if positive results were obtained, based on the factors of the case. In this case the investigator at the scene did not see a need to have the vehicle and driveway processed at that time.
Defense went on to ask, "could it be possible that the Fluorescein reaction that was detected at the crime scene was from blood deposited two years ago"? Again the answer was yes, it's possible. Defense was trying to discredit the value of the Fluorescein. However, DNA was obtained, and DNA is subject to degrading under certain circumstances. So Fluorescein did lead to the recovery of DNA, but now it's the job of the DNA Expert to the life expectancy of DNA at a crime scene.
Based on my years of experience, I am not so concerned about the arguments made against the Fluorescein process, because the same arguments will be the same if any other method such as Blue Star, or Luminol were to be used. However, the important factor is that the process lead to the location of the DNA! Once the DNA is found, it would be up to the DNA Experts to answer the question like, how long will DNA remain viable (how long will it take for DNA to degrade under whatever conditions).
In theory the detectable elements in the latent blood do not evaporate away, nor will degrade in time (i.e. iron or rust after the iron oxidizes). So Fluorescein can cause a reaction, if there is something there to react to, for an indefinite duration of time, however environmental factors can shorten this time period. Constant exposure to water can dilute the amounts of concentration of the latent blood. So the bottom-line is that Fluorescein works, and is one of the major tools available in the arsenal of latent blood detection.
There are several ways that one can obtain Fluorescein for forensic law enforcement applications. Many labs reflux or manufacture their own versions of Fluoreseine. RC Forensics is the main manufacturer and patent holder of the product . Abacus Diagnostics, Inc. is a licensed distributor, and sales Fluoreseine under the product name HemaScein™, and does make direct sales to any law enforcement agency . Abacus Diagnostics has several kits that can include the following: Fluorescein based confirmatory test, ABACard® HemaTrace® human blood testing kits, evidence collection swabs, ABA fine mist sprayers, and a powerful LED Forensic Light Source. Another vendor that stocks HemaScein™ is Arrowhead Forensics (www.arrowheadforensics.com 800.953.3274).
I would like to thank my friends Robert Cheeseman, Forensic consultant, (RC Forensic) and W.D. "Doc" Moseley, Police Reserve Officer (San Bernardino Police Department) for their assistance in reviewing and editing this paper. I would further like to thank Robert Goss, Property Manager and Debbie Greenlea, Forensic Supervisor; with the San Bernardino Police Department, who have given me support and encouragement in the researching and writing of this paper.
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