|
ABSTRACT:
Fluorescein was considered to be the reagent with
the greatest potential in the detection of latent bloodstains in the California Criminalistics Institute (CCI)
Maucieri and Monk study in 1991. [1] The purpose of this study was to develop and improve the fluorescein
technique into a practical field system for the detection of latent bloodstains.
Current Material Safety Data Sheets (MSDS) state
fluorescein to be no more hazardous than luminol, presently in use by many investigative organizations. Also,
fluorescein has a twenty-year history in the medical field of ophthalmology and is Food and Drug Administration
(FDA) approved for clinical application in retinal and choroidal angiography. [2]
Unfortunately, unlike luminol's single reagent
application, fluorescin requires an application of itself followed by hydrogen peroxide (H2O2). This double reagent application can be problematic,
especially on vertical, non-porous surfaces, resulting in bloodstain pattern distortion due to reagent running.
A commercial thickener was used to overcome this problem, affording crime scene photographers greater opportunity
to document the bloodstain patterns as evidence.
INTRODUCTION:
During the 1980's, the Environmental Protection
Agency (EPA) became more strict with respect to toxic and / or mutagenic reagents utilized in the workplace.
Many reagents used cavalierly in the past were placed under greater scrutiny, resulting in the enforcement of more
stringent safety guidelines. Luminol has been erroneously listed as banned for use in California in some
literature. The current MSDS lists both luminol and fluorescein as "possible carcinogenic" hence;
no such ban is in place on these reagents at this time.
The literature lists fluorescein as the reagent
with the greatest promise to replace luminol. [3] Fluorescein has been used in the clinical setting since the 1940's,
and more recently has been the angiography diagnostic mainstay for vascular ophthalmic disorders requiring FDA
approval for clinical use. [2] This clinical heritage and FDA sanction may put to rest the safety issue with
respect to the exposure to fluorescein.
The need for a safe, reliable bloodstain enhancement
technique can be very valuable, and the application of the fluorescein technique should be limited to latent stains
only. The two factors which most directly bear on the effective use of this technique are either the blood
concentration which has been so diluted it can no longer be seen by any other means, and / or the surface on which
the stain is located lacks adequate color contrast differential. If any neat bloodstains are found, adequate
samples should be taken for further serological testing before the fluorescein technique is employed. Due
to the high alkaline nature of the fluorescein technique, any humoral studies (ie; ABO, anti-human, secretor status)
may not be possible afterwards.
The substrate surface texture the stain is located
upon also plays an important role. Porous versus non-porous surfaces, and vertical versus horizontal factors
are important to the success of this technique, or can prove to be problematic as in the case of a non-porous substrate
on a vertical surface. To alleviate this problem, a commercial thickener, Keltrol RD, or xanthan gum, an
exocellular heteropolysaccharide produced via fermentation, was employed. [4] This was added to the fluorescein
diluent which helped to slow the dispersion, or distortion of the bloodstain pattern after the fluorescein was
applied, preserving the bloodstain pattern for documentation. With the increased viscosity, it became necessary
to utilize a power-sprayer to produce even and consistent coverage for the target area. Previously,
a hand spray pump bottle was adequate for this, and still is for the application of hydrogen peroxide. However,
sufficient pressure is not possible for the more viscous reagent application for the fluorescein. A Low Pressure,
High Volume (LPHV) spray gun was attained because of its capacity to cope with the higher viscosity and ability
to deliver the reagent in a controlled, contained and detailed manner.
In its reduced (colorless) state, fluorescin has a very short shelf life. The literature calls for usage within 48 hours.
However, even after 24 hours the change back to its oxidation (colored) state, fluorescein was
sufficient to cause problems with background fluorescence. [1][3] Even though it is possible to prepare fluorescein
in the laboratory and transport it to the crime scene, the fresher the fluorescin reagent is the more effective
it will be, hence the need for field preparation. For this technique to be truly field worthy, a premeasured
and aloquated kit format was adopted. This required a more simplified preparation procedure, which should
be within the skill level of crime scene technicians. Minimizing the equipment and skill required to prepare
the fluorescin reagent is critical to its success in the field. Again, I would like to stress that this technique
is to be utilized after all other tests of interest have been performed and its only application is on latent bloodstains.
MATERIALS AND METHODS:
I Reagents and Equipment
Fluorescein
Hydrogen Peroxide (H2O2)
NaOH
Hot Plate / Stirrer
Zinc Powder
Glassware
Keltrol RD
II Delivery Devices
Hand pump spray bottles
LPHV power spray gun
III Documentation Equipment
Alternate light source, 450nm filter and wand
35mm SLR camera
Orange barrier filter (Nikon #056) (Note:
it has been suggested that a yellow filter may allow greater visualization
Video camcorder
Tripods, etc.
IV Personal Safety Equipment
Particle facemask
Orange eye goggles
Latex gloves and lab coat
Reagent
Preparation
Fluorescein Reagent
1) Make up 10% sodium hydroxide solution
(10.0 grams sodium hydroxide in 100ml deionized water). A stock quantity of this solution may be kept on-hand
provided it is stored in a nalgene type container. Storage of strong bases in glass containers is not recommended.
2) Weigh out 1.0 gram of fluorescein and
allow it to dissolve in 100ml of the 10% sodium hydroxide stock, using a 250ml Erlenmeyer flask with a stir bar
to facilitate dissolving.
3) While the fluorescein is dissolving completely
into solution, weigh out 10.0 grams of zinc powder.
4) Place the Erlenmeyer flask with
the fluorescein / sodium hydroxide solution on the hot plate / stirrer. Stir and heat gently. Add the
10.0 grams of zinc powder to this solution, and continue to heat and stir to a gentle boil. (Note:
the zinc will not dissolve) This solution will lose most of its color at this time. Allow the
solution to cool and the zinc to settle.
5) Decant the solution off being careful
to exclude any of the undissolved zinc. Make a 1:20 dilution (1 part (50ml) decanted solution with 19 parts (950ml)
deionized water / dissolved commercial thickener) and mix thoroughly. This will be the fluorescin reagent solution.
6) When the commercial thickener is
used, approximately (5.0 grams Keltrol RD per 1000ml DI water) in the total volume of the fluorescin solution is
adequate. However, the Keltrol RD dissolves slowly and should be rehydrated in advance. This may be
stored frozen, then thawed and used in the fluorescin's diluent.
Hydrogen Peroxide Solution
1) Make up a 10% hydrogen peroxide
solution (1 part (100ml) 30% hydrogen peroxide with 2 parts (200ml) deionized water). A stock solution of
this may be kept in an opaque bottle and refrigerated.
2) At the time of use, place 300ml
in the second spray bottle.
Application Procedure
1) Take spray bottle #1 (the 1:20 fluorescin
dilution) and spray the area of interest. Mist the area with a fine, uniform spray from a distance of 12"-18".
Make two applications in this fashion, being careful not to make the misting so heavy as to cause the reagent to
run. Allow a few seconds for the color to develop.
2) Take spray bottle #2 (10% hydrogen
peroxide solution) and apply in the same manner described above.
3) Observing standard safety practices
for using UV light, be sure that all personnel wear safety goggles.
4) Illuminate the area of interest
with near UV light (450nm).
Application of the fluorescin dilution (bottle
#1) on the targeted area of bloodstain will develop a yellow colorization within a few seconds if blood is present.
At this stage, the bloodstain will be apparent and some background colorization may be apparent. However,
the application of the hydrogen peroxide dilution (bottle #2) will help to reduce the background and false positive
reaction, thus clarifying the bloodstain pattern. Using a near UV light source the bloodstained areas will
fluoresce when illuminated.
QUALITY CONTROL:
If possible, simultaneously perform a strongly
reactive control, weakly reactive control and negative control during routine examination. Also, either simulate
the substrate of the area of interest, or utilize an actual portion of the area of interest, properly labeled.
LIMITATIONS AND PRECAUTIONS:
The purpose of this technique is to enhance the
appearance of latent bloodstain patterns. It will not differentiate the type, or source of the bloodstain,
and can yield false positive results with certain substances (ie; Fe, Cu or soil (bacterial contamination)).
Two separate spray applications are necessary for
this technique. A light and even misting yields the most successful results. However, non-porous vertical
surfaces are susceptible to reagent running, which can distort or destroy any bloodstain patterns, and use of the
commercial thickener is recommended in all circumstances.
Two major safety precautions exist with this technique.
First, the usage of 10% sodium hydroxide (strong base pH 13.5) is extremely caustic and dangerous to work with.
Also, storage of strong bases in glass containers is not recommended. Second, UV light can be very hazardous
if the proper precautions are not implemented and safety goggles employed.
RESULTS:
The goal of this effort will be to focus on the
following areas:
1) To simplify the chemical procedure
for reducing fluorescein to fluorescin.
2) To verify aspects of Monk's results
with fluorescein respective to sensitivity, false positive (specificity) and establish optimum working dilutions.
3) To resolve the reagent running problem,
hence expanding the documentation window.
By attaining the fore mentioned goals, it is hoped
that the fluorescein technique will be appreciated as a truly field worthy and practical procedure remaining within
the scope and skill levels of crime scene technicians and investigators, hence resulting in an improvement in documentation.
1) Simplification procedure
of fluorescein to fluorescin
In previous literature, fluorescein (oxidized color
state) was reduced to fluorescin (reduced colorless state) via heating the fluorescein (1.0 gram) in 10% NaOH (100ml)
with 10.0 grams zinc powder. This was originally accomplished by the use of a refluxing column, so as not
to boil away the solution. This task maybe routine for chemists, but would not be conducive to field production.
The elimination of the refluxing apparatus and only bringing the solution to a boil does not appear to have reduced
the effectiveness of the resulting fluorescin solution. Once established, this adaptation was used as the
standard technique utilized in all subsequent experiments, which is previously delineanated in Materials and Methods.
To determine if any sensitivity would be lost due
to the adoption of the simplification procedure, the following experiment was conducted. Utilizing a common
serial blood dilution (ranging from 1:1000 to 1:105,000) two racks of test tubes (12x75mm) were set up. Each
tube received equal portions (approx. 30microliters) of blood dilution, fluorescin (1:3) and H2O2 (10%). Rack A utilized fluorescin reduced via the
refluxing apparatus, and rack B utilized fluorescin reduced via the adapted simplification technique. Negative
controls (blanks) were used with each rack in which the blood dilution was substituted with deionized water.
Both racks were illuminated with a Wood's lamp (long wave UV). A plus / minus system was utilized to grade
each tube for fluorescence. Equal results were achieved with each blood dilution and blank, establishing
the sensitivity was positive at 1:105,000 for both reduction techniques.
2) Specificity (cross reactivity
/ false positives)
The purpose of this experiment was to determine
what other common substances may react with fluorescin yielding false positive results. All of the tested
items are typically found in residential settings and may be mistaken for bloods by either coloration (red / brown),
or reaction to the fluorescin reagent (fluorescence). (Table 1)
Table 1
Stained Items
Fluorescence
Inherent UV (at 450nm)
|
Control Blood
|
-
|
+
|
|
Saliva
|
-
|
-
|
|
Coffee
|
+
|
-
|
|
Tea
|
-
|
-
|
|
Grass Stains
|
-
|
-
|
|
Soil*
|
-
|
-
|
|
Chocolate
|
+
|
-
|
|
Cola
|
+
|
-
|
|
Strawberry Jelly
|
+
|
-
|
|
Ketchup
|
+
|
-
|
|
Beet Juice
|
+
|
+
|
|
Horseradish
|
+
|
+
|
|
Cherry-strawberry Juice
|
+
|
-
|
|
Cherry-cranberry Juice
|
+
|
-
|
|
Also Tested:
|
|
|
|
Urine
|
-
|
+
|
|
Steel
|
-
|
-
|
|
Steel (with rust)
|
-
|
+
|
|
Aluminum
|
-
|
-
|
|
Copper
|
-
|
+
|
· This was positive in the literature, however this soil yielded
a Negative result. Soils may vary greatly from one location to another.
Work Dilutions
Two separate experiments were conducted during this study
to determine the optimum fluorescin dilution to use. The first experiment was similar to the procedure simplification
experiment (equal portions in test tubes) of testing varying dilutions of fluorescin ranging from 1:3 to 1:150
against blood dilutions ranging from 1:6000 to 1:105,000. (Table 2) Grading the reaction with zero
to four-plus system resulted in the following:
Table 2
Blood
Dilutions x 1000
|
6
|
12
|
15
|
24
|
36
|
48
|
75
|
96
|
105
|
blank
Fluorescin Dilutions
|
|
4
|
4
|
3
|
3
|
2
|
1
|
1
|
+
|
+
|
0
1:3
|
|
4
|
3
|
3
|
2
|
2
|
1
|
1
|
+
|
+
|
0
1:50
|
|
2
|
2
|
1
|
1
|
1
|
+
|
+
|
0
|
0
|
0
1:100
|
|
1
|
1
|
+
|
0
|
0
|
0
|
0
|
0
|
0
|
0
1:150
|
The second experiment utilized varying blood dilution
absorbed into strips of blotter paper. Based on the results of the previous experiment, the fluorescin dilution
range was narrowed to a scope of 1:3 to 1:50. Due to the different substrate, the blood dilution was changed
to a scope of 1:10 to 1:24,000. (Table 3) Due to the difficulty in subjectively judging the reaction
of the fluorescein on this substrate, a simple plus / minus grading system was employed:
Table 3
Blood Dilutions
|
1:1000
|
1500
|
2000
|
2500
|
3000
|
6000
|
12000
|
24000
|
Fluorescin Dilutions
|
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
1:3
|
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
1:10
|
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
1:20
|
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
1:30
|
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
1:40
|
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
-
|
1:50
|
3) Documentation Improvement
Because of the need to apply two reagents (fluorescin
dilution / hydrogen peroxide), reagent running was cited as a significant shortcoming to this technique.[1]
If this problem could be overcome, it would improve documentation for court presentation. A commercial thickener,
such as that used in "guaranteed one-coat" house paints, Keltrol RD was utilized.[4] For this product
to be useful in this technique, three criteria must be met; 1) It must be able to tolerate the high
pH of the fluorescin without detrimental effects to its thickening properties, 2) It must not
cross-react with the fluorescin, or cause any detrimental effects on the fluorescin's properties, and, 3)
It must be capable of being applied in an even and consistent manner which is viscous enough to prevent reagent
running and establish bloodstain pattern stabilization long enough for documentation. All horizontal and
vertical glass panes were similarly bloodstained (Figure 2). The before (Figure 2) and after depiction (Figure
3) demonstrates the problematic nature of double reagent systems (fluorescin / H2O2), on a non-porous substrate (glass panes). The two glass panes on the right in Figure
3 illustrate the improvement with the Keltrol RD additive.
Figure 1
Figure 2
Same glass panes after one minute
of being processed with
fluorescin without Keltrol RD (left) and with Keltrol RD (right)
Early on, the manufacturer (Kelco) assured me that
pH should not be a problem, since its product was derived via fermentation and endured other rigorous demands in
that process.
The cross-reactivity would be known immediately
as soon as the Keltrol and fluorescin were mixed together, and a blood sensitivity gradient test was conducted
without any negative side effect. In fact, the sensitivity was slightly greater with the Keltrol, probably
due to stabilizing the reagents on the bloodstain, allowing the reagents more time to react. However, it
was apparent immediately that the hand pump delivery system would not be capable of facilitating any viscosity
necessary to alleviate the reagent running problem. Hence, a power spray delivery device would be necessary
to satisfy the last (third) criterium. A 1.0% stock solution of Keltrol RD was utilized and later diluted
to the desired viscosity (0.5%) and used in the fluorescin's diluent.
Keltrol RD Concentration
Delivery System
< 0.2% / volume .............................................. Hand pump spray bottle
0.3% - 0.6% / volume .......................................Power
spray device
DISCUSSIONS AND CONCLUSIONS:
Considering that this technique would be one of
the last procedures performed at a crime scene, it is possible for the crime scene investigators to notify the
laboratory to prepare the fluorescin reagent and transport it to the crime scene. However, protection of
the fluorescin reagent via darkened opaque nalgene bottles will be necessary. Exposure of the reduced (colorless
state) fluorescin to sunlight or any UV light source may prematurely oxidize the fluorescin back to the oxidized
(colored state) fluorescein, hence undermining the effectiveness of this technique.
The "Kit" concept yields greater latitude
to the investigators in two aspects. First, with investigative organizations, which have geographically large
jurisdictions, and transporting reagent from the laboratory to the crime scene is not practical for time or distance
reasons. Secondly, the simplified reduction procedure requires less equipment and skill level to prepare;
hence more personnel will be capable of performing this task, perhaps yielding better utilization of crime scene
staff. Conversely, this technique can only be applied once to a target area. Each time a bloodstain
is sprayed, to some degree dispersion of the pattern (distortion) will occur. Also, the background fluorescence
will be very high on target areas, which have been previously treated.
Once it was realized that a power spray device
would be necessary to facilitate the increased viscosity of the Keltrol RD, a power spray manufacturer was consulted.
Originally, I had thought the airless power spray would suffice because it would not emit the NaOH fumes except
during the actual spraying, however the spray gun manufacturer advised me that only the most powerful guns could
accommodate the necessary viscosity, and the high pressure (approx. 3000psi) at which these guns operate would
not be conducive to small areas or detailed application. A Low Pressure (approx. 30psi), High Volume (LPHV)
gun was suggested, due to its ability to cope with the Keltrol's RD viscosity, and its ability to deliver the reagent
in a confined and detailed manner, reducing the chance of over spray. Noxious NaOH fumes are emitted all
the time the gun is powered, hence the need for the particle face masks. Due to the one shot nature
of this technique and the potential of destroying evidence, it is advised that only personnel with adequate training
and experience should be permitted to employ this technique.
The documentational improvement afforded by this
technique via Keltrol RD (xanthan gum), has further broadened the scope from investigative more toward evidentiary
in nature. [4] This should result in better court presentation and ultimately a clearer understanding by
the trier of fact. This may also broaden or expand the usage of this technique. Previously, this technique
would not have been thought appropriate or effective, in distinguishing class characteristics of shoeprints on
various substrates, including carpeting. Visualizing a totally latent foot trail, may lead the investigator
to additional prints on a more favorable substrate, perhaps yielding to individualizing characteristics.
Preliminarily, results currently under study have
demonstrated this technique to be at least as sensitive as luminol if not slightly better, although the literature
empirically favors the sensitivity of luminol. [5] The effects of environmental conditions to which biological
samples might be exposed by this technique, either from chemical or UV light, appear to be constrained and predictable
according to current DNA analysis technology. [6] Preliminary DNA analysis via PCR has also been successful
after employment of the fluorescin treatment. Both of these topics are slated for further study in the near
future.
Documentation Guidelines
The fluorescin reaction can be documented with
still photography or by video camcorder. The scene should be photographed prior to the fluorescin application.
Examine the substrate with the suspected bloodstain for any native fluorescence and document any results by photography.
Test the fluorescin reagent on sample blood and check for the proper reaction. Apply the reagent on a like
substrate, check for cross-reactivity and document the results. The reaction can be visualized and photographed
with and without the use of an orange barrier filter. Unlike luminol photography, the scene does not need
to be completely darkened for the reaction to be visualized and photographed. Some ambient light in the scene
will aid the photographer / investigator in later orienting the scene and its contents in the resulting documentation,
and alleviates the need for fill flash photography.
Photographic documentation of the fluorescin reaction
is best accomplished with a tripod-stationed 35mm camera with the aperture set at F/8 using an orange or yellow
barrier filter and color print film (EI400). Vary the exposure times bracketing between 5 and 30 seconds,
depending upon the lighting conditions at the scene. Photographs may also be taken utilizing the aperture-priority
automatic function of the camera, resulting in quality photographs.
The fluorescin reaction can occur for several minutes
before the bloodstain pattern begins to degrade and background fluorescence becomes problematic. This allows
ample time for the photographers to vary their exposures, and document the scene with and without the orange barrier
filter. (Note: a yellow barrier filter may also yield favorable results)
References
1. Monk, J.W., "Fluorescent Bloodstain
Detection - A Replacement For Luminol", MSc project report to CCI, 1991
2. Reichel, Elias M.D.; Puliafito,
Carmen M.D.,"Indocyanine Green (ICG) Angiography In the Diagnosis and Treatment of Choroidal Neovascularization",
Clinical Modules, New England Eye Center, January 1994.
3. Maucieri, L.; Monk, J., "Enhancement
of Faint and Dilute Bloodstains With Fluorescence Reagents", California Association of Criminalists, Summer
1992.
4. Xanthan Gum Booklet, "Xanthan Gum
- Natural Biogum for Scientific Water Control", Fifth Ed., Kelco Company, San Diego, CA., June 1994.
Technical Bulletin DB-31, "Preservatives for
Kelco Polymers Used in Industrial Applications", Kelco Company, San Diego, CA., November 1986.
Technical Bulletin DB-38, "KELTROL RD Xanthan
Gum in Food, Personal Care and Pharmaceuticals", Kelco Company, San Diego, CA., June 1994
For information on these bulletins, please contact:
Chicago, IL 800 535-2687
Clark, NJ 800 535-4141
5. Identification News, "Sensitivity
Comparison of Blood Enhancement Techniques", August 1985, pp 10.
6. Farley M.; Harrington J., Forensic DNA Technology, chapter 5, pp 75.
|
