I.            INTRODUCTION

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.

II.            MATERIALS AND EQUIPMENT

            A.            CHEMICALS

1.       Fluorescein (95% dye content, e.g., Aldrich F245-6)

2.       Zinc, powdered and mossy

3.       Ethanol (99.5%, denatured)

4.       Glacial acetic acid

5.       Hydrogen peroxide (H₂O₂ )

6.       Sodium hydroxide

7.       Distilled water

            B.            EQUIPMENT

1.       Small bottles and vials

2.       Chromistâ type sprayers

3.       Larger plastic garden type sprayers

III.            SAFETY

Read 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.

 IV.           ETHANOLIC REAGENT PREPARATION: 

A.                STOCK SOLUTION:  In a small bottle combine:

0.1 g fluorescein

20 mls ethanol

2.0 g powdered zinc

1 ml glacial acetic acid

The 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

fluorescin solution may be stored over fresh zinc (mossy zinc), but yields optimum results when freshly prepared. 

B.        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.

C.              H₂O₂  OVERSPRAY:  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.

V.            AQUEOUS REAGENT PREPARATION:

A.      STOCK SOLUTION:  In a small bottle combine:

0.1 g fluorescein

2.0 g powdered zinc

20 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.

B.            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). 

C.            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).

A.            SENSITIVITY AND CONTRAST CHECK:

Dilute 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

in 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).

B.            CHECK FOR NATIVE FLUORESCENCE:

After 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.

C.            SPRAYING TECHNIQUES:

Use 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 appropriate. 

1.            Lightly mist the target with the reduced fluorescin solution.

2.                   Overspray by misting with the 3% H₂O₂ ethanol solution.  This can be re-applied as needed.  3% aqueous H₂O₂ can also be used.

D.            LIGHT SOURCE:

Use 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.

E.                 PHOTODOCUMENTATION:

Photograph 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. 

1. 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. 

2.. 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. 

F.            NOTES:

1. It is important to visually examine surfaces before spraying. Fluorescein spraying is a presumptive searching technique and is subject to false positives similar to luminol or phenolphthalein (e.g., copper, hypochlorite). As the title of this procedure states, this procedure is to be used for latent bloodstains. It is useful for examining areas or items where bloodstains are suspected, where stains are indistinct, or where cleanup or coverup of bloodstains is suspected.

2. 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.

3. 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.

4. 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.

5. 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.

6. A fluorescent ruler may be used when taking photographs.

7. Oxygen can also re-oxidize the fluorescin-heme complex and you will see some fluorescence before you overspray with H₂O₂.  

     8. Other presumptive blood testing reagents may be applied to a substrate that   has been treated with fluorescein. Fluorescein sprayed materials are suitable for PCR DNA analysis.

VII.            INTERPRETATION:

Fluorescein 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 analyses.

VIII.            REFERENCES:

1.            “Detection of Latent Blood – A New Fluorescein Formulation” Boan, T., DiBenedetto, J., Marie, C., IABPA Presentation, Houston, TX 11/98

2.            “Enhancement of Faint and Dilute Bloodstains with Fluorescence Reagents”, Maucieri, L., and Monk, J., CAC News, Spring 1992, pp. 13 – 20

3.            “Fluorescein and Latent Blood Detection”, Cheeseman, R., and Durgin, G., AAFS 50th Anniversary Meeting, 2/98

4.            San Diego Sheriff’s Crime Laboratory – “Fluorescein Spraying for Latent Blood, Reagent Preparation Protocol”

5.            “Fluorescein:  A Tool for Visualizing Latent Blood”, Marie, C., IABPA Presentation, Harrisburg, PA 10/02

6.            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, Aug. 1983.