Chemical Enhancement of Fingerprints in Blood: An Evaluation of Methods, EFFECTS ON DNA, and Assessment of Chemical Hazards

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

Introduction

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.


On dark surfaces, none of these color-producing reagent stains could be guaranteed to produce detectable prints. Attempts to use luminol had reportedly been of limited success since the brief chemiluminescence created was weak, hard to photograph, and failed to resolve fine ridge detail. Recent authors have recommended new techniques for the development enhancement of faint blood prints on various surfaces. In a 1995 review of techniques, John Neuner, North Carolina SBI, suggested leuco crystal violet [4,4',4"methylidynestris(N,N dimethyl aniline)] (a reduced form of crystal violet) as a reagent for developing dark-colored prints on light-colored backgrounds, and merbromin for developing prints on dark backgrounds, exploiting the merbromin-blood complex' fluorescing properties(1). He gave no processing details or evaluation. Bodziak recommended leuco crystal violet for the development of shoeprints in blood (2). Maucieri and Monk recommended fluorescin (the reduced form of fluorescein) as a potential candidate(3). Cheeseman and DiMeo successfully applied fluorescin to bloody fingerprints in a viscous medium to reduce running(4). Although it was not mentioned in the literature, DFO (1,1 diazafluoren-1-one), with its demonstrated sensitivity to amino acids in normal skin secretions, was thought to have potential for blood prints. Everse and Menzel suggested the use of merbromin (mercurochrome) to develop fluorescing prints on non-porous surfaces(5). It must be remembered that any blood-bearing surface - friction ridge skin, tool, weapon, glove or shoe - can be of interest to the crime scene examiner, so techniques developed for enhancement of blood-residue fingerprints can be of use in many other applications.


It was thought that a side-by-side comparison of the available techniques on typical target surfaces would be of value. Their sensitivity (using both serial dilution and sequential-touch methods of producing concentration gradients) and applicability to a variety of surfaces could be readily compared. In addition, interference or visualization problems could be evaluated. Also, since many blood bearing "fingerprint" surfaces may also be subjected to DNA analysis, interferences with current DNA methods could be evaluated using known human blood and subjecting it to the harshest chemical exposure from each method.


Experimental
A folded paper towel "touch pad" was dampened with fresh whole human blood (anti-coagulant added, but no preservative) until touching it just produced an even film of blood on the tips of the fingers. Three fingers (index, middle, ring), were touched simultaneously on the touch pad and then immediately touched repeatedly to the target surface. The use of three fingers gives some control over finger-to-finger touch variations without having to create another test target. Test targets were a variety of porous, non-porous and semi-porous surfaces typical of the surfaces on which bloody prints are found in casework. The targets consisted of: porous: white typing paper, black construction paper, brown bag paper, bare wood (balsa); semi-porous: white contact (shelf) paper, textured gypsum board painted with a semi-gloss latex wall paint, weathered fiberglass panel; and non-porous: smooth-surface glass bottles, plastic soft drink bottles, and aluminum soft drink cans. The used or weathered targets were cleaned in hot water and allowed to dry before prints were placed on them. All blood prints were placed on all targets by the same person on the same day.

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.
In addition, whole human blood was serially diluted in DI water from 1:10 to 1:1,000,000. Single drops of these dilutions were then placed on white typing paper and allowed to dry at ~30°C for 4 hours before test reagent solutions were applied.


The following reagent solutions were made for these tests:
Ninhydrin:
0.6% solution in acetone
Amido black (AB):
0.2% solution in methanol/glacial acetic acid (9:1)
Methanol/glacial acetic acid (9:1) rinse
DFO:
Stock Solution:
0.5% diaza-fluoren-1-one in methanol/acetic acid (9:1)
Working Solution: 60ml stock solution + 10ml 2-propanol + 50ml acetone + 50ml xylene + 830ml petroleum ether
Fluorescin:
Stock Solution: Dissolve 1g fluorescein in 10%
NaOH with 10g Zinc metal. Boil until clear and
pale in color
Working Solution: 5% stock solution in water
Leuco Crystal Violet (LCV):
Dissolve 10gm 5-sulfosalicylic acid (Aldrich 24700-6)
3.7g sodium acetate
1g Leuco crystal violet (Aldrich 21921-5) in 500 ml 3%
hydrogen peroxide
Merbromin:
Stock Solution: 0.45g merbromin in (Aldrich 19959-1) 100ml ethanol, 15ml formic acid, 10g mossy zinc. Reflux until clear and pale in color.
Working Solution A: 10ml stock/30ml acetone
Working Solution B: 10% hydrogen peroxide/acetone (1:9)

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:
Ninhydrin: Applied by immersion (dipping) the target into the reagent solution for 5s, then allowing excess to drain away. Air dried and then developed at room temperature and humidity for 7 days prior to reading, or developed in 70°C/70% humidity for 2 hrs.
DFO: Applied by immersion, two applications, allowed to air dry between. Heated at 85°C in a dry oven and evaluated using 532nm laser.
Amido Black: Applied using a squeeze bottle, rinse applied with squeeze bottle, then cold tap water rinse.
Leuco crystal violet: Applied using a fine-mist aerosol applicator in a high-draft fume hood. Allowed to develop for 30 seconds, then rinsed with cold tap water.
Fluorescin: Applied using a fine-mist aerosol applicator in a high-draft fume hood. Allowed to develop at room temperature and then examined using UV and laser light sources.
Merbromin: Applied using a fine-mist aerosol applicator, (both solutions) allowed to dry at room temperature, and then examined using a laser light source.
Upon completion of the chemical treatment of the test targets, the resulting prints were evaluated visually by two experienced latent print examiners for intensity (detectable color against the background) and clarity (readability of developed ridge detail). For color-producing reagents: (ninhydrin, LCV, AB), the evaluations were carried out in normal room light. For fluorescing reagents (merbromin, DFO, fluorescin) the evaluations were carried out using illumination of appropriate wavelength and using suitable barrier filters when needed. The prints were rated according to the number of the sequential touch that demonstrated detectable color on fluorescence and identifiable ridge detail. Each target, then would have a two digit rating, such as 4/3 or 6/6. This rating takes into account background coloration or reactivity as well as loss of clarity by solvent action.


Evaluations of the sequential touch targets were repeated at 10 day intervals, up to 40 days after treatment. Results are presented in Tables 1 and 2, in the next section.
The serial dilution targets were rated in a similar fashion with each target being rated on a scale of 0 to 5+ for intensity (color) against a white background. See Tables 1 and 2.


Since the possible source of the blood associated with a bloody fingerprint or shoe impression could be an important issue, the impact of these six reagents on the ability to successfully type DNA from bloodstains using 14 PCR-based markers was examined. Given that the blood associated with an evidence impression can be quite limited, PCR-based markers were chosen since they are one of the most sensitive typing techniques available. In addition, successful amplification of DNA with several of these primer sets and characteristic human typing profiles are considered to be sufficient analytical information to establish that the analyzed DNA is of human /primate origin.


Bloodstains were prepared by adding 30ml of whole, human blood (anti-coagulant added, no preservative) to clean cotton swabs. These stains were allowed to dry for 24 hours at room temperature and two bloodstained swabs were each dipped into one of the following reagents: merbromin, amido black, fluorescin, LCV, DFO, or ninhydrin reagents. Three similarly prepared bloodstains were used as control samples and were not treated with the fingerprint reagents. Following this treatment, the bloodstains were maintained for 11 days at room temperature and then stored frozen (-15°C) for 20 days.


The DNA was extracted from all bloodstains by digestion with Proteinase K, purification with phenol/chloroform and concentration through microfiltration. The quality of the resulting DNA was evaluated by running the extracted DNA in a 1% agarose gel. With the exception of the samples treated with merbromin or ninhydrin, no significant degradation was noted in the DNA obtained from the treated bloodstains or the untreated control stains. The DNA from the bloodstains treated with merbromin or ninhydrin displayed high molecular weight DNA with an associated faint smear of degraded DNA. Following the agarose gel evaluation, the DNA was prepared for amplification in the following PCR Amplification Kits from PE Applied Biosystems (Foster City, CA.): DQA1/PolymarkerÔ, AmpliFLP D1S80Ô, AmpFLSTRÔ GreenI and AmpFLSTRÔ Blue. Using the protocol provided by the manufacturer, 2ng to 5ng of DNA (in a 20ul fixed volume solution containing 160ng of BSA) was added to the amplification cocktails for the DQA1/Polymarker and D1S80 loci. The amplified DNA from these 7 loci (D1S80, DQA1, LDLR, GYPA, HBGG, D7S8 and GC) was then typed by either immobilized probe typing strips or polyacrylamide gel electrophoresis. Following the manufacturer's protocol, approximately 1.0 ng of DNA was added to the AmpFLSTRÔ GreenI and Blue amplification cocktails. The amplified DNA from these 7 loci (Green1: Amelogenin, THO1, TPOX, CSF1PO and Blue: D3S1358, VWA, FGA) was typed using capillary electrophoresis on an ABI Prism 310 Genetic Analyzer. The resulting data was analyzed using GeneScanÔ /GenotyperÔ software.

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

Reagent

Dilution

Rating (Intensity)

LCV (freshly made)

1:10

+4

 

1:100

+3

 

1:1000

+2

 

1:10,000

+1

 

1:100,000

-

 

1:1,000,000

-

Comparison Test on Painted Gypsum Board: 6/6 (Intensity/Clarity)

 

Amido Black

1:10

+5

 

1:100

+4

 

1:1000

+3

 

1:10,000

+2

 

1:100,000

+1

 

1:1,000,000

-

Comparison Test on Painted Gypsum Board: 6/6 (Intensity/Clarity)

 

Ninhydrin

1:10

+5

(Evaluated after 5

1:100

+4

days at room

1:1000

+3

Temperature and

1:10,000

+2

Humidity)

1:100,000

+1

 

1:1,000,000

-

Comparison Test on Painted Gypsum Board: 5/4 (Intensity/Clarity)

 

 

Table 4: Fluorescing Reagents, Serial Dilution: Blood on Black Paper

 

Merbomin

UV (254 nm)

Laser (532 nm)

1:10

+2

+3

1:100

+1

+2

1:1000

0

+1

1:10,000

0

0

1:100,000

0

0

1:1,000,000

0

0

Comparison on

6/5

4/4

Painted Gypsum   Board

No Background Fluorescence

High Background Fluorescence

   

Fluorescin

Absorption

No fluorescence

1:10

+1

1:100

0(bkgrnd)

1:1000

0(bkgrnd)

1:10,000

0(bkgrnd)

1:100,000

0(bkgrnd)

1:1,000,000

0(bkgrnd)

High background

Old (60days) fluorescin on black paper with UV (360nm): UV: 3/0

 

 

LCV (dark red fluorescence with 1.2W dye laser @ 532nm)

 

 

 

1:10

Absorption

No fluorescence

1:100

 

+3

1:1000

 

+2

1:10,000

 

+1

1:100,000

 

0

1:1,000,000

 

0

 



Table 4: Results: Color Reagents, Sequential Touch

LCV: Targets Prepared 6/19/97.  Stock Solution Prepared 6/19/97

 

Target

Result

@ 1 day

Re-eval

@ 40 days

Result

@ 10 days

Re-eval

@ 28 days

Result

@ 30 days

Re-eval

@ 8 days

 

Brown paper

 

  5/4

 

  5/5

 

  5/4

 

  5/4

 

  5/3

 

  4/3

 

White  paper

 

  6/5

 

  6/6

 

  7/4

 

  4/4

 

  4/4

 

  5/4

 

Gypsum Board

 

  6/6

 

  6/4

 

  6/6

 

  6/6

 

  6/4

 

  6/4

 

Wood (raw)

 

  6/6

 

  6/6

 

  5/2

 

  5/5

 

  -

 

  -

 

Contact paper*

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/5

 

Fiberglass*

 

  6/6

 

  5/5

 

  5/4

 

  5/4

 

  3/2

 

  3/3

 

Plastic bottle*

 

  8/8

 

  8/8

 

  5/5

 

  5/5

 

  4/4

 

  5/5

 

Glass Bottle*

 

  7/7

 

  7/7

 

  7/7

 

  7/7

 

  4/4

 

  4/4

 

Aluminum can*

 

  3/3

 

  4/4

 

  4/4

 

  5/5

 

  5/5

 

  6/6

 

Gypsum board (sooted)*

 

  -

 

  -

 

  -

 

  -

 

  6/6

 

  6/6

 

Aluminum can (sooted)*

 

  -

 

  -

 

  -

 

  -

 

  2/2

 

  -

 

 

*  Water rinse @ 30 seconds.

   High background develops upon exposure to room light


Table 1  Amido Black: Targets Prepared 6/19/97.
Solution prepared 6/10/97

Target Result@ 1 day Re-eval@ 40 days Result@ 10 days Re-eval@ 28 days Result@ 30 days Re-eval@ 8 days

Target

Result

@ 1 day

Re-eval

@ 40 days

Result

@ 10 days

Re-eval

@ 28 days

Result

@ 30 days

Re-eval

@ 8 days

 

Brown Paper*

 

  5/5

 

  5/5

 

  3/3

 

  5/5

 

  3/3

 

  5/5

 

White Paper*

 

  3/3

 

  5/3

 

  6/6

 

  6/6

 

  4/4

 

  4/4

 

Gypsum Board

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

Wood (raw)*

 

  4/4

 

  4/4

 

  3/3

 

  4/4

 

  -

 

  -

 

Contact Paper

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

Fiberglass*

 

  5/5

 

  5/5

 

  4/4

 

  5/4

 

  4/4

 

  5/5

 

Plastic Bottle

 

  7/7

 

  7/7

 

  6/6

 

  6/6

 

  5/5

 

  7/7

 

Glass Bottle

 

  6/6

 

  7/7

 

  7/7

 

  7/7

 

  8/8

 

  8/8

 

Aluminum Can

 

  6/6

 

  6/6

 

  7/7

 

  7/7

 

  6/6

 

  6/6

*Significant background after rinse with MeOH/HAc and water


Table 1/Ninhydrin

Ninhydrin: Targets prepared 6/19/97, Solution Prepared 6/10/97
Target Result@ 1 day Re-eval@ 40 days Result@ 10 days Re-eval@ 28 days Result@ 30 days Re-eval@ 8 days

Target

Result

@ 1 day

Re-eval

@ 40 days

Result

@ 10 days

Re-eval

@ 28 days

Result

@ 30 days

Re-eval

@ 8 days

 

Brown Paper*

 

  5/5

 

  5/5

 

  3/3

 

  5/4

 

  5/4

 

  5/4

 

White Paper*

 

  6/4

 

  6/6

 

  5/4

 

  5/4

 

  6/6

 

  6/6

 

Gypsum Board

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

Wood (raw)*

 

  3/3

 

  4/4

 

  5/4

 

  5/4

 

  -

 

  -

 

Contact Paper

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

 

  6/6

*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.

 

Result

@ day 1>

 

6/20/98

 

Wood

 

 

  3/3

 

Black paper

 

 

  -

 

Brown paper

 

 

  5/5

 

White paper

 

 

  6/4

 

Shelf paper

 

 

  6/6

 

Dry wall

 

 

  6/6

 

 

Table 2: Results: Fluorescing Reagents, Sequential Touch
Merbromin: Targets Prepared 6/19/97. Stock Solution Prepared 6/12/97

Target Result@ 1 day Result@ 10 days Re-eval@ 28 days Result@ 30 days Re-eval@ 8 days Re-eval@ 40 days

Target

Result

@ 1 day

Result

@ 10 days

Re-eval

@ 28 days

Result

@ 30 days

Re-eval

@ 8 days

Re-eval

@ 40 days

 

Brown Paper*

 

  5/4

 

  4/4

 

  4/4

 

  4/4

 

  3/3

 

  5/5

 

White Paper*

 

  6/5

 

  1/1

 

  0/0

 

  3/2

 

  0/0

 

  0/0

 

Gypsum Board

 

  5/3

 

  0/0

 

  0/0

 

  4/3

 

  4/3

 

  0/0

 

Wood (raw)*

 

  6/0

 

  6/0

 

  6/0

 

  -

 

  -

 

  4/0

 

Contact Paper

 

  5/5

 

  6/6

 

  6/6

 

  6/6

 

  5/4

 

  4/3

 

Black Paper

 

  3/3

 

  -

 

  -

 

  -

 

  -

 

  6/6

 

Gypsum Board

 (sooted)

 

  -

 

  -

 

  -

 

  4/3

 

  0/0

 

  -

 

Aluminum Can

 (sooted)

 

  -

 

  -

 

  -

 

  3/3

 

  -

 

  -


Fluorescin: Targets prepared 6/19/97. Stock solution prepared 6/19/97.

 

Brown paper*

 

3/0

 

0/0

 

0/0

 

  0/0

 

  4/0

 

  4/0

 

  0/0

 

White paper*

 

5/3

 

3/0

 

2/0

 

  3/1

 

  4/4

 

  4/2

 

  3/2

 

Gypsum Board*

 

5/0

 

6/0

 

0/0

 

  5/4

 

  3/2

 

  5/2

 

  3/2

 

Wood (raw)*

 

5/0

 

6/0

 

6/0

 

  6/0

 

  -

 

  -

 

  -

 

Contact paper

 

-

 

-

 

-

 

  -

 

  -

 

  -

 

  -

 

Black Paper

 

3/0

 

-

 

-

 

  -

 

  -

 

  -

 

  -

 

*High background fluorescence

 

         

Results:

  DFO

Evaluate @ 1day

 

Brown Paper*

 

 -2/2

 

No fluorescence in blood, absorption

 

White Paper*

 

 -1/1

 

No fluorescence in blood, absorption

 

Gypsum Board*

 

 -5/5

 

No fluorescence in blood, absorption, very high background fluorescence

 

Wood (raw)*

 

 -5/5

 

No fluorescence in blood, absorption, very high background fluorescence

 

Contact Paper

 

 -2/2

 

No fluorescence in blood, absorption

 

Black Paper

 

 0/0

 

No fluorescence in blood, absorption

 

          Detail not visible due to absorption of blood against
background.

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 multiplexes.
In summary, clear-cut typing results were obtained for all 14 PCR-based markers from the DNA extracted from the bloodstains treated with each of these 6 reagents. Since the bloodstains in this study were made on absorbent cotton swabs, this study did not examine the effect of the mechanical manipulations on the possible loss of sample. This would be an important factor to take into consideration when dealing with a limited amount of blood on a non-absorbent surface (e.g. plastic/glass ). Any significant loss of samples will negatively impact any typing results.

References
1. Neuner, John K., "Enhancement of Blood Contaminated Impression Evidence," Presented at the IAAI Training Conference, Costa Mesa, CA, July 1995.

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".

See Also:
Jaret, Yvan; Heriau, Michel; and Donche, Alain, "Transfer of Bloody Fingerprints," J. Forensic Ident, 47(1), 1997 p.p. 38-41.

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)

--General--

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--

--Toxicological 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.

Carcinogenic/Mutagenic/Reproductive Properties

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.

Non-Carcinogenic Properties

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.
The Aldrich MSDS (1997) added a caveat to the Toxicology information section, stating:

"To the best of our knowledge, the chemical, physical, and toxicological properties have not been thoroughly investigated."

--Other Hazards--

If involved in a fire, LCV can emit toxic fumes of carbon monoxide, carbon dioxide, and nitrogen oxides (Aldrich, 1997).

--Exposure Thresholds/Guidelines--

There are no regulatory or recommended airborne or skin absorption thresholds established.

Summary

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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