Fluorescein is useful as a presumptive searching reagent for latent bloodstains. Whole blood drops apparently quench the fluorescence and fluorescein is not recommended for this type of stain. However, fine bloodstains and blood smears will fluoresce when treated with fluorescein. Two formulations are included in this procedure. The alcohol-based formulation is useful for vertical or slick surfaces because it will not run as readily as the aqueous formulation. It is also a better selection when the recovery of fine detail is important. There is less background intereference with the alcohol formulation, but it is not as bright as the aqueous formulation. The aqueous formulation may be selected for large searches on porous and/or horizontal surfaces where fine detail is not critical.
MATERIALS AND EQUIPMENT
Fluorescein (95% dye content, e.g., Aldrich F245-6)
Zinc, powdered and mossy
Ethanol (99.5%, denatured)
Glacial acetic acid
Hydrogen peroxide (H2O2 )
Larger plastic garden type sprayers
the appropriate MSDS’s. Zinc is
considered a combustible solid. It reacts with acid, water or moisture in the
air. If the zinc particle is small
enough, heat of reaction may be enough to self-ignite when with organic
materials. Ethanol is a flammable
liquid with a flash point of 55 oF.
If you are working in an enclosed space, respiratory protection is
recommended. Good ventilation is
recommended at all times. Glacial
acetic acid is a corrosive substance and is also considered a flammable liquid
with a flashpoint of 109 oF. Hydrogen
peroxide is very irritating to eyes, skin and respiratory system.
Hydrogen peroxide is an oxidizer, which may make organic substances more
flammable on contact.
ETHANOLIC REAGENT PREPARATION:
SOLUTION: In a small bottle
g powdered zinc
ml glacial acetic acid
solution will become colorless soon after mixing.
Let this reduction reaction go for at least 30 minutes.
Warming the vial with your hand or placing the vial in warm water will
accelerate the process. Hydrogen is
generated and may be vented occasionally by loosening the cap. This stock
solution may be stored over fresh zinc (mossy zinc), but yields optimum results
when freshly prepared.
WORKING SOLUTION: The
fluorescin spraying solution is made by decanting or pipetting stock fluorescin
solution off the zinc and then diluting it 1 : 100 in ethanol (e.g. add 1 ml
stock solution to 99 mls ethanol). The
working solution is best used within a few hours.
It should be discarded when the spraying is done.
The hydrogen peroxide overspray is prepared by diluting 30% H2O2
to 3% H2O2 in ethanol.
(i.e. 10 mls 30% H2O2 diluted up to 100 mls with
ethanol) If you are not concerned
about reagents running, you can also overspray with aqueous 3% H2O2
purchased over the counter.
AQUEOUS REAGENT PREPARATION:
STOCK SOLUTION: In a small
g powdered zinc
mls distilled water.
Add 1.0 g sodium hydroxide pellets and mix thoroughly.
The reaction is moderately exothermic upon addition of the sodium
hydroxide. Warming the solution
with your hand, or in a small beaker of warm water, will help drive the
reduction of fluorescein to fluorescin.
WORKING SOLUTION: Decant or pipette the reduced fluorescin solution off
the zinc and dilute the solution in 1:100 in distilled water
(i.e. 1 ml stock to 99 mls distilled water).
OVERSPRAY SOLUTION: The
overspray solution is 3% H2O2 which can be purchased over
the counter or diluted from 30% H2O2 (1:10 in either
distilled water or ethanol).
SENSITIVITY AND CONTRAST CHECK:
whole blood in distilled water minimally covering a range of from 1/100 to
1/400,000. Pipette about 20uls of
each dilution onto white filter paper. These
can be prepared, air-dried and kept
a freezer until needed. Each time
fluorescin/ H2O2 sprays are used they should be first
tested on the blood dilution series. Note
the sensitivity and contrast (background).
CHECK FOR NATIVE FLUORESCENCE:
a visual examination, check the area to be sprayed with your light source for
native fluorescence. Mark these
areas to avoid confusion after spraying. The
area or item to be sprayed should be documented before spraying.
a TLC type sprayer (e.g., Chromist sprayer) for small and/or fine detail work.
Use an approximately ½-gallon to one-gallon plastic garden pump type
sprayer for searching and for larger areas. Other non-metallic sprayer as
Lightly mist the target with the reduced fluorescin solution.
Overspray by misting with the 3% H2O2 ethanol
solution. This can be re-applied as
needed. 3% aqueous H2O2
can also be used.
an alternate light source at 445 nm and orange or yellow barrier filters
(goggles). Different irradiation
wavelengths and barrier filters may be used as indicated depending on the
substrate to be illuminated.
areas of interest using an orange (or yellow) barrier filter (i.e., Nikon 52).
Use the same barrier filter for photography as you use for visualization.
You may want to use a tripod.
35 mm suggested parameters: 400ASA, f8. Set
zoom and focus with lights on. Expose
in darkness for 10, 20 and 30 seconds. Without
changing the zoom or moving the camera, take an additional photo with visible
light. Note that fluorescent areas
sometimes photograph better when the light source is not too intense.
Digital camera: Turn
the flash off on the digital camera. Use
an orange (or yellow) barrier filter in front of the lens.
Again, photograph with and without lights on.
Concentrated blood may quench the fluorescence.
We see fluorescence around the edges of concentrated bloodstains.
This technique is meant for latent blood – not patent blood.
Examine the substrate with the alternate light source before spraying and
mark any “native” fluorescence. If
possible, test a small part of the substrate by staining it with a known
dilution of blood, allowing it to dry and then spraying.
This will give you a positive control for the substrate in question.
It is better to apply very light mists of both sprays.
More can be added if needed. The
finer the spray, the better the detail. Items
may be re-sprayed.
Zinc reduces fluorescein to fluorescin and it becomes colorless.
The brightest form of fluorescin is thought to be the dianion, which is
the predominant species at about pH 9. (The
aqueous formulation is prepared in NaOH for this reason.)
H2O2 reoxidizes the fluorescin-heme complex and
promotes the fluorescence.
A fluorescent ruler may be used when taking photographs.
Oxygen can also re-oxidize the fluorescin-heme complex and you will see
some fluorescence before you overspray with H2O2.
is a presumptive test for blood. It
is useful in the detection of patterns of older, indistinct or latent
bloodstains and in detecting the residue of blood remaining after a stain has
been cleaned. The appearance of a
greenish-white fluorescence indicates the possible presence of blood.
The appearance of fluorescence alone should not be interpreted as
positive proof of blood. Chemical
oxidants, such as those found in bleach, are potential sources of false positive
reactions. The sensitivity of this
test on blood dilutions on white filter paper is approximately 1x104.
However, the sensitivity of this reagent varies depending on the
substrate tested, how much blood was deposited,
and how the blood was deposited.
Spraying with fluorescein does not affect retrieval of DNA for PCR based
“Detection of Latent Blood – A New Fluorescein Formulation” Boan,
T., DiBenedetto, J., Marie, C., IABPA Presentation, Houston, TX 11/98
“Enhancement of Faint and Dilute Bloodstains with Fluorescence
Reagents”, Maucieri, L., and Monk, J., CAC News, Spring 1992, pp. 13 – 20
“Fluorescein and Latent Blood Detection”, Cheeseman, R., and Durgin,
G., AAFS 50th Anniversary Meeting, 2/98
San Diego Sheriff’s Crime Laboratory – “Fluorescein Spraying for
Latent Blood, Reagent Preparation Protocol”
“Fluorescein: A Tool for
Visualizing Latent Blood”, Marie, C., IABPA Presentation, Harrisburg, PA 10/02
Specht, W., “The Chemiluminescence of Hemin:
An Aid for Finding and Rcognizing Blood Stains Important for Forensic
Purposes”, Sourcebook in Forensic
Serology, Immunology, and Biochemistry, Unit IX, pp. 67-69, published by the NIJ,