Dr. J.D. DeHaan, J.D. Clark, T.F. Spear, R. Oswalt, S.S. Barney, CA Department of Justice, Bureau of Forensic Services, Sacramento, California
Blood is commonly encountered as a transfer medium for fingerprints at crime scenes. Sometimes, the residue retains enough color to allow it to be photographed directly, but, more often, the residue is so faint that its color (and thereby its contrast) is so slight that ordinary light photography is ineffective except on transparent or highly reflective sources. The advent of tunable-wavelength light sources (Polilight et al) allowed the latent print examiner to pick an examination wavelength at which the residual hemoglobin most strongly absorbs, thereby increasing its potential contrast, especially on surfaces that reflect (or even fluoresce) at that wavelength. Chemical methods for the enhancement of residual blood fingerprints, have been successfully used for years. Leucomalachite green, amido black, and ninhydrin chemically react with components in blood to form a dark-colored dye complex and have all been used successfully on light-colored or transparent surfaces. Leucomalachite green and ninhydrin have low background colors but are unsuitable for non-porous surfaces as they run off, and either distort the print or fail to react before detail can be photographed. Amido black is very sensitive and works well on non-porous surfaces but its high background color (light to medium blue) compromises contrast on porous surfaces from which the stain cannot be removed by rinsing.
It had been previously found by the authors during the
preparation of test targets for latent print training exercises
that sequential touches of a surface provides a convenient,
reproducible gradient of concentration of the transfer medium.
No matter what the medium is: blood, grease, or natural skin
oils, each touch removes a percentage of the medium, leaving
less on the skin for the next touch (until it is replenished).
Multiple sequential touches, thus, produce a series of target
transfers, each having less medium than the one just before it.
A single target then offers a range of concentrations for a
single chemical treatment to react with. Multiple duplicate
targets were prepared for each reagent to be tested as
appropriate. For instance, ninhydrin and DFO were to be used
only on porous or semi-porous targets. Tests would be conducted
1 day, 10 days and 30 days after prints were deposited. The
blood-printed targets were allowed to dry for approximately 18
hours at room temperature (68°F, 22°C) and humidity (~40%)
until first processing.
Each target was treated according to the best established
method for each reagent (or according to recommendations of
authors discussing the newer techniques), as follows:
Results The results of color-developing reagents on
sequential touch targets are presented in Table 1. The results
of fluorescing reagents on sequential touch targets are
presented in Table 2.
Table 3: Results: Color Reagents
Serial Dilution: Blood on White Paper
Comparison Test on Painted Gypsum Board: 6/6 (Intensity/Clarity)
Comparison Test on Painted Gypsum Board: 6/6 (Intensity/Clarity)
Comparison Test on Painted Gypsum Board: 5/4 (Intensity/Clarity)
Table 4: Fluorescing Reagents, Serial Dilution: Blood on Black Paper
Old (60days) fluorescin on black paper with UV (360nm): UV: 3/0
LCV (dark red fluorescence with 1.2W dye laser @ 532nm)
Table 4: Results: Color Reagents, Sequential Touch
LCV: Targets Prepared 6/19/97. Stock Solution Prepared 6/19/97
* Water rinse @ 30 seconds.High background develops upon exposure to room light
Target Result@ 1 day Re-eval@ 40 days Result@ 10 days Re-eval@ 28 days Result@ 30 days Re-eval@ 8 days
*Significant background after rinse with MeOH/HAc and water
Ninhydrin: Targets prepared 6/19/97, Solution Prepared
*Developed @70°C/70% humidity for 1 hr. prior to evaluation just used on one set for 6/20/97. The table above shows values not used with a heat accelerant. Those values are listed below.
Table 2: Results: Fluorescing Reagents, Sequential Touch
Target Result@ 1 day Result@ 10 days Re-eval@ 28 days Result@ 30 days Re-eval@ 8 days Re-eval@ 40 days
*High background fluorescence
Detail not visible due to absorption of blood against
EFFECTS ON DNA ANALYSIS
All 12 treated bloodstains (each duplicate set of stains
treated with a different reagent) were successfully typed in all
14 loci. The types were readily interpretable and the same types
were obtained with the treated and untreated bloodstains. The
typing profiles obtained with the treated bloodstains were
comparable to the typing profiles of the untreated bloodstains.
The only noticeable differences between the treated and
untreated bloodstains involved the bloodstains dipped in the
merbromin and ninhydrin reagents. The differences which were
observed were: (1) a faint product band for the DQA1 locus for
the ninhydrin treated bloodstains and (2) the larger loci in the
AmpFLSTRÔ GreenI and AmpFLSTRÔ Blue (CSF1PO and FGA,
respectively) showed a minor reduction in signal intensity. The
intensity differences noted in the CSF1PO and FGA loci resulted
in a slight imbalance across the loci in each of the two
2. Bodziak, William, "Use of leuco crystal violet to enhance shoe prints in blood." Forensic Science International, 82, 1996 p.p. 45-52.
3. Maucieri, Louis A. and Monk, Jamie W. "Enhancement of Faint and Dilute Bloodstains with Fluorescence Reagents. (CAC News), Summer 1992.
4. Cheeseman, Rob and DiMeo, Lisa Allyn, "Fluorescein as a Field-Worthy Latent Bloodstain Detection System." "J. Forensic Ident., 45(6) 1995, p. p. 631-646.
5. Everse, K.E. and Menzel, E.R., "Blood print detection by fluorescence". "Center for Forensic Studies, Texas Tech University, Lubbock, Texas".
Chemical Hazard Information
This section will look at the individual chemicals used in the LCV formulation and summarize the available information regarding known or potential hazards.
It is important to note that many chemicals, especially a good percentage of those used in forensics, have not been thoroughly (sometimes even minimally) investigated for their toxicological effects or other health and safety impacts. In some cases, inferences may be drawn from anecdotal or associated literature regarding potential health and safety impacts.
Leuco Crystal Violet (LCV)
As noted earlier, LCV is the reduced form of crystal violet (CV), also known as gentian violet. The description of the LCV method indicates LCV is oxidized to crystal violet, with a corresponding color change from clear to purple-violet. Therefore, both forms of the chemical are to be considered in the hazard assessment.
Available information does not indicate any epidemiological or other human studies have been conducted on the health and safety impacts of LCV/CV. There are some genetic, cellular, and animal studies on the toxicological properties of CV, and no known studies of that type on LCV.
--Physical & Chemical Properties--
LCV and CV are organic chemical compounds that belong to the aromatic amine family. This family contains chemicals such as aniline, benzidine, o-tolidine, o- and p-Toluidine, and others (Scott, 1983). Many of these aromatic amines are either known, suspected, or potential carcinogens (cancer causing), mutagens (disrupt the genetic code), and teratogens (cause malformed offspring due to the inheritance of disrupted genetic code).
Various chemicals in this family are also known to act as skin sensitizers, respiratory irritants, liver toxins, and acute poisons via adverse reactions with red blood cells leading to the formation of methemoglobinaemia.
Generally speaking, the aromatic amines can readily be absorbed into the body through the skin, lungs, and gastrointestinal tract. Therefore, all three exposure pathways are efficient means of entry for these compounds (Scott, 1983). It is particularly important to emphasize the fact that nearly all the aromatic amines are lipid soluble, with the concomitant ability to easily penetrate the human skin. The majority of other chemical compounds do not share this specific ability.
Additionally, the bacterial flora and enzymatic actions in the human gut and liver appear to assist in making some, otherwise larger and/or less reactive aromatic amines, smaller, potentially more toxic, and more easily absorbed by the stomach and intestines. In the process, a greater toxic dose of the aromatic amine metabolites is taken into the body and systemically distributed. This specific metabolic activity has been described for a subset of the aromatic amine chemicals, namely the benzidine-based dyestuffs and congeners (Boiteau, 1983; Bell, Breslin, and Lemen, 1980).
While LCV and CV are members of the aromatic amine family, the literature on health impacts and hazards of that large group of compounds does not identify, nor exclude them, as chemicals of concern. Again, lack of comprehensive, chemical specific health studies prohibit those types of observations and conclusions. This is due in large measure to the relatively marginal use of LCV/CV in industry and the resulting lack of attention by health professionals.
Several animal studies have shown CV capable of producing various adverse reproductive effects ranging from developmental abnormalities to death of the fetus. CV was administered orally (ingestion exposure pathway) in relatively low to moderate doses - i.e. 7 to 100 mg/kg body weight. The animals used in the studies were rats and rabbits (Micromedex (RTECS), 1997).
CV mutagenicity studies performed using various bacteria and mammalian cell types
The California Safe Drinking Water and Toxic Enforcement Act of 1986, also known as Proposition 65, lists "benzidine-based dyes" as "Chemicals Known to the State to Cause Cancer or Reproductive Toxicity. The listing was made on October 1, 1992. The listing was added to the section containing carcinogenic substances. Clarification regarding the scope of that listing was requested of the California Environmental Protection Agency - Office of Environmental Health Hazard Assessment, the agency responsible for developing and updating the listing. It is unclear at this time if LCV/CV are included in the aforementioned listing.
Minimal human data on the non-carcinogenic health effects of LCV or CV was available.
Several animal studies have been performed to characterize the acute toxicity of CV. Both non-lethality and lethality studies have been undertaken on mice, guinea pigs, dogs, cats, rats, and rabbits (Micromedex (RTECS), 1997). The non-lethality studies reported various non-carcinogenic health effects ranging from pulmonary edema to gastrointestinal distress and weight loss. Dosing was performed orally with initial (lowest concentration) observations reported from 20 mg to 1 gm/kg body weight of animal.
Lethality studies used to determine the concentration of CV that would kill 50% of the tested animal populations (referred to LD50) reported oral and injected dose LD50's ranging from 5 to 420 mg/kg. These concentrations, by a pesticide labeling standard, would be characterized as 'highly to moderately toxic'. Animal specie tested and route of exposure appear to influence the reported LD50 value.
Human skin irritation tests reported positive results (Micromedex (RTECS), 1997). The Aldrich Chemical Company - Material Safety Data Sheet (1997) notes LCV as a skin, eye, and respiratory system irritant, requiring appropriate engineering and/or personal protective equipment exposure control measures.
The LCV Material Safety Data Sheet (MSDS) lists two LD50
values, both in excess of 5000 mg/kg, indicating relative low
toxicity via oral and skin exposure dosing experiments on mice.
These values are significantly higher than reported for CV.
"To the best of our knowledge, the chemical, physical, and toxicological properties have not been thoroughly investigated."
If involved in a fire, LCV can emit toxic fumes of carbon monoxide, carbon dioxide, and nitrogen oxides (Aldrich, 1997).
There are no regulatory or recommended airborne or skin absorption thresholds established.
It is critically important to reiterate that the forensic community has an ethical and legal obligation to protect the health of both its own forensic professionals, and the public it serves. To this end, the methods, procedures, and investigative tools used to process evidence should be selected and employed in such a manner as to maximize the health and safety of the aforementioned groups.
The use of leuco crystal violet for enhancing latent blood impressions appears to be an efficacious tool from a forensic analysis perspective. The described LCV method employs a liquid formulation that can be applied to target substrates via spraying, dipping, or immersion.
However, while the body of health and safety data is far from complete, the available toxicological information indicates that LCV has some hazards which makes its' use at crime scenes or other uncontrolled environments problematic to both law enforcement (forensic professionals) and the public.
Of chief concern are the potential exposure hazards from LCV itself. The three main exposure pathways - i.e. inhalation, skin absorption, and ingestion, are all viable routes of entry given the proposed method of application and use. LCV is an aromatic amine and an analogue of the aniline compounds and benzidine-based dyes. Given the general chemical properties associated with the aromatic amines, it is likely the skin, lungs, and gastrointestinal tract readily absorb LCV. Further, many of the benzidine-based dyes are metabolized in the human body into benzidine, which is known to cause cancer. However, it is unclear, based upon available information, whether LCV/CV is environmentally degraded or metabolized via that pathway.
Very little data could be found regarding the environmental fate of LCV/CV. Environmental persistence, degradation pathways, and data on biologically active degradation products are not available. Related information notes that LCV/CV is light sensitive. One study observed enhanced cellular genetic toxicity with light incubated cultures (Levin, Lovely, Klekowski, 1982). The effects of time, temperature, humidity, light, pH, microorganisms and various environmental chemical matrices on the stability and intrinsic toxicity of LCV/CV are unknown.
The few animal and cellular toxicological studies that have been performed on LCV point toward an ability to cause mutagenicity, other adverse reproductive health effects, and cancer. Criteria established by the Registry of Toxic effects of Chemical Substances (RTECS) qualify LCV to be considered a potential carcinogen.
Toxicity studies indicate that LCV/CV is 'highly to moderately' toxic, depending upon the route of exposure, animal specie tested, and the toxic endpoint observed (i.e. lethality vs. specific non-lethal endpoints). It should be noted the reported lethal toxicity values varied significantly in the literature reviewed. LCV is also considered a skin, eye, and respiratory system irritant.
Given prudent personal hygiene practices and handling precautions, the moderate to low toxicity or mild irritation hazards associated with using 5-sulphosalicylic acid, sodium acetate, and hydrogen peroxide (at the proposed concentrations) can be effectively managed. Of course, mixing and diluting these compounds from technical grade or 'neat' stocks can lead to potential exposures. In the case of hydrogen peroxide, it is presumed that only a 3% stock solution will be procured, handled and used in the preparation of the LCV formulation.
Recommendations on Use
1. I do not recommend field use of the leuco crystal violet formulation in other than very tightly controlled applications. While the scientific evidence is not overwhelming and in some instances equivocal, there does appear to be both carcinogenic and non-carcinogenic health end-points that can result from exposure to LCV/CV. Further, all exposure pathways are potential means of entry. Uptake appears to be relatively quick, with metabolic processes potentially increasing the intrinsic toxicity of the LCV/CV. Law enforcement and the public could be both acutely and chronically exposed to LCV/CV.
2. If field use of LCV/CV is contemplated, the following actions are be taken:
A. The entity providing forensic support at the crime scene should undertake a risk management evaluation of the issues. Specifically, upper management should assess the evidentiary worth and forensic value of using LCV/CV, versus, the potential health effects and long term liability from applying chemical compounds considered problematic.
B. Fully inform the law enforcement agency in control of the crime scene about the materials to be used and the potential short and long-term hazards involved to both forensic professionals and the public. Liability issues should be completely resolved, and clearly understood by all involved entities. A written and signed letter of notification (or other suitable documentation) should formalize the process.
C. At the crime scene, the target substrate receiving the LCV formulation should be clearly identified. All practical measures should be taken to reduce the amount of substrate tested to an absolute minimum.
D. In the absence of any data regarding effective means for removal of LCV from applied substrates, the target substrate(s) should be cutout or otherwise removed from those crime scenes where rehabitation of the building/structure is likely. The LCV formulation can then be applied in an outdoor or better controlled environment where improved ventilation is possible and residual contamination minimized.
E. Aerosolized LCV formulation should be kept to an absolute minimum due to the lack of ventilation control and air flow at crime scenes. Off target spread of aerosolized LCV and the subsequent exposure hazards clearly discourage spray application and should not be used. Other application methods need to be employed and/or researched.
F. All personnel applying the formulation and subsequently handling the evidence (target substrate) should wear appropriate personal protective equipment. This would include disposable, chemical protective body coverings, gloves and boots; and full-face supplied air or air-purifying respirator. The respiratory protection is used when the formulation is applied and the evidence handled in the 'wet' or 'fresh' state. If air-purifying respiratory protection is selected, canisters or chemical cartridges capable of removing amines, organic and aromatic compounds, and aerosols within the HEPA (High Efficiency Particulate Air) classification, are to be used.
G. All personal protective and evidence handling equipment which is visibly contaminated or reasonably anticipated to be contaminated with the applied formulation, is to be bagged and disposed of as hazardous waste, or subsequently cleaned in a controlled laboratory environment.
H. Good personal hygiene, including strict adherence to hand washing and the avoidance of hand-to-mouth activities during the use of LCV is required.
I. Efforts are to be made to limit the number of individuals applying the formulation, handling the evidence after application, or otherwise potentially exposed to the LCV formulation.
J. The law enforcement agency in charge of the crime scene is to post the scene, clearly providing notification of the type and kind of chemicals used in the processing of evidence and the general hazards associated with their exposure.
3. Application of the LCV formulation can be accomplished in a controlled forensic laboratory environment, providing adequate local exhaust ventilation (i.e. chemical fume hood) and personal hygiene measures are employed.
The formulation is to be applied in a properly operating fume hood, with average hood inflow face velocities not less than 150 linear feet per minute. The 150 lfm is the minimum regulatory hood face velocity when working with carcinogenic materials. Airflow into the hood is to be smooth with no eddy or backwash currents.
Personnel applying the materials are to utilize chemical protective gloves, aprons or other body coverings, and protective eyewear such as goggles and/or faceshields.
Again, judicious use of the formulation is strongly recommended. Spraying is discouraged. Other application methods which generate minimal amounts of fugitive aerosols are encouraged.
Hand washing and the avoidance of hand-to-mouth activities are to be stringently enforced.
The written Chemical Hygiene Plan for the laboratory should be updated to reflect the use of potential carcinogenic materials and the control measures used to minimize personnel exposure.
4. Should the use of LCV evolve beyond the research and development stage into impression evidence casework, a consensus of law enforcement agencies should enjoin the National Institute of Justice, National Institute of Health, or some other governmental body to sponsor detailed toxicological research studies. The studies would scientifically evaluate the scope and breadth of health and safety effects from using LCV/CV.
5. Research should be performed on the means, methods, and
efficacy of removing and/or decontaminating target substrates
that have been applied with the LCV formulation.
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