Frequently Asked Questions on Hemascein®
How does the recovery of DNA from Hemascein compare with luminol containing reagents such as Bluestar?
“Hemascein also performed significantly better than Bluestar Magnum, Lumiscene and Lumiscene Ultra. Hemascein preserved DNA to a greater extent than the rest of the reagents. Lumiscene significantly decreased the success of DNA profiling success” [Citation].
“For the effects on DNA tests, high peak was obtained on the electroferogram compared to the leucomalachite green method or the luminol method and DNA detection wasn’t affected.”
[Citation]
Figure 4.40: Bar chart showing the relative DNA profiling success from bloodstains treated with the various reagents
(The manufacturer of lumiscene has expressed to us that it disputes the above published results.)
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Please click on images above to see a larger view
[Citation]
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[Citation]
[Citation]
“ More than 10 years after the original homicidal event occurred, investigators retrieved the clothing from evidence and applied Hemascein to it. They noticed small reaction in small areas on the clothing. The lab was able to produce a full profile of human blood. Subsequently, the blood was matched through a CODIS hit to ….” Bloodstains As Evidence: A Field Manual (Page 121)
Which peer reviewed sources may I cite to show the differences in DNA recovery?
Illuminating Latent Blood
Hemascein Implementation DNA Recovery Vermont State Police
Effectiveness of New Bloodstain Preliminary Examination Reagent
Validation of novel latent bloodstain detection kit Hemascein, Korea
Does ABAfix affect DNA?
No.
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Ambient Light or Complete Darkness
Does Hemascein require complete darkness like luminol containing reagents such as Bluestar?
Hemascein does not require complete darkness. Cases have been investigated in street night light [Citation]. This makes viewing the bloodstain in context with the crime scene easier. “In terms of applying the different reagent to a crime scene, Bluestar Magnum,Lumiscene, Lumiscene Ultra and Grodsky are all luminol based reagents.
Therefore, they need darkness to view the reaction. Increasing the ambient light
levels decreases the ability to see the chemiluminesence. A Hemascein reaction
with blood, on the other hand, can be viewed when ambient light levels are not
entirely dark. This makes movement around the crime scene easier. Also,
photographing the scene is easier because the reaction can be viewed in relation to the crime scene without overlaying images as done in luminol photography (section1.4.7). Also, because of the long reaction time of the Hemascein reaction, repeat photography can be carried out. This is better than repeated applications of luminol as there is no further dilution of the sample. [Citation]
“We could search and take pictures under relatively bright Environment. “[Citation]
“Hemascein™ does not need darkness condition to detect latent blood stains. Very useful where unable to cut out environmental lights.”[Citation]
“Crime Scene personnel applied Hemascein® to the pathway leading to the area where they found the shoe imprints with an outsole similar to the impressions in the garage (See Figures CS4-6 through CS4-8). Numerous partial footwear impressions became visible after application of Hemascein®.” Bloodstains As Evidence: A Field Manual (page 118)
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How long Hemascein illuminates for me to take pictures so that I am not constrained to 30 seconds as in luminol and do not have to continue diluting my samples ?
“Hemascein had the longest reaction time with Lumiscene Ultra and Grodsky having the longest reaction times for the luminol based reagents. From the above table it can be seen that Bluestar Magnum has the shortest reaction length. ”
[Citation] .
It is recommended that before spraying the reagent(s), use your alternate light source on the target area as a negative control to know of any potential background fluorescence that you may need to account for later. Also start taking pictures the moment you spray every few seconds and you may continue taking pictures upto 10 minutes depending upon the substrate and case in hand.
Click on image above to see a large view of Figures 4.17 through 4.21
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Is the shelf life of Hemascein better than luminol reagents?
“In the activated form, the shelf life of the luminol based reagents is significantly shorter than the shelf life of the Hemascein working solution. Grodsky, Lumiscene and Lumiscene Ultra had the lowest shelf life at four hours after the working solution was made. Blue star Magnum had a longer shelf life of 24 hours. However, the working solution of Hemascein provided was by far the longest stability, being stable for up to 7 months”
[Citation]
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Grodsky |
Lumiscene |
Lumiscene Ultra |
Bluestar
Magnum |
Hemascein |
Self life Activated |
4hrs |
24hrs |
4hrs |
4hrs |
7months max |
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Is Hemascein more sensitive than Luminol containing reagents (Grodsky, Lumiscene, Bluestar) and Leucomalachite Green?
“In regards to the verification of detection sensitivity, it was superior than the leucomalachite green method or the luminol method.”
[Citation]
“Both luminol and Bluestar® produced an intense chemiluminescence-based light emission with liquid blood samples during the first minute of the test. This was followed by a rapid decay of light intensity. In contrast, HemasceinTM produced a nearly constant fluorescence-based light emission with intensities that equaled or surpassed those of Bluestar® and luminol after the initial one minute time period.” [Citation]
“..in one case, it lasted 18 months on fingerprints found on a door” Bloodstains As Evidence: A Field Manual (Page 123)
Is Sensitivity of Hemascein higher on diluted bloodstains ? Why?
Yes. Concentrated blood quenches the fluorescence as the excitation light does not fully penetrate the full width of a highly concentrated sample. It is also believed that if a sample is too concentrated, the emission light may be reabsorbed by the fluorophore attenuating the fluorescence signal at shorter wavelengths, resulting in apparent red shifted emission. Hemascein® is for latent blood and not for patent blood and therefore diluted samples will appear easier to see due to more light being produced. Hemascein is most effective on diluted and aged bloodstains that are most commonly encountered when considering latent bloodstains.
Is Sensitivity of Hemascein higher on burnt bloods?
Yes. [Citation]
I just put whole blood on a surface and can see it with my eyes. But it does not light up with Hemascein?
Hemascein is to reveal invisible blood and not for whole blood that is visible without it. On whole blood and concentrated bloodstains, you may see fluorescence only around its edges. Concentrated blood quenches the fluorescence as the excitation light does not fully penetrate the full width of a highly concentrated sample. It is also believed that if a sample is too concentrated, the emission light may be reabsorbed by the fluorophore attenuating the fluorescence signal at shorter wavelengths, resulting in apparent red shifted emission.
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Is Hemascein a discovery tool or an enhancement tool?
Hemascein is a discovery tool to identify bloodstains that are not visible. Neither Hemascein nor any of the Luminol reagents should be used if the stain is already visible. In such case, you should try Hematrace or send it through DNA analysis.
I was able to see fingerprints and footwear impression with Hemascein. Therefore why can’t I use it as an enhancement technique in addition to the discovery?
It is true that during discovery, Hemascein sometimes may bring out the image of latent bloody fingerprints or shoewear impressions. But Hemascein should not be used as a method for enhancement on bloodstains that are already visible (the benefit of getting such enhancement of bloodstains during discovery of latent bloodstain is unique to the Hemascein when compared to luminol).
If you can see it and want to enhance the bloodstain, use a bloodstain dye such as Ashley’s Reagent, Coomassie Blue, Leuco Crystal Violet and Hungarian Red to enhance it.
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Length of Time to Read Results
How long should you read Hemascein results after? How long after you should not read the results?
It is recommended that before spraying the reagent(s), use your alternate light source on the target area and take pictures as a negative control to know of any potential background fluorescence that you may need to account for later. Start taking pictures the moment you spray every few seconds.You may continue taking as many pictures until 10 minutes depending upon the substrate and case in hand. It is recommended that a portion of substrate without blood on it can be treated with just the reagent to observe potential background staining prior to applying Hemascein.
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I understand that Hemascein may give background in certain situations?
First, background is not unique to hemascein but is also present in luminol known as “background chemiluminesence effect”. The reason one may not see such luminol background chemiluminesence development with luminol as the reaction may not last more than 30 seconds to allow its development to be visible. As an example, Proescher and Moody in Journal of laboratory and clinical medicine in 1939 had found that HCl increased background lumiscence in luminol. So did Catello, Alvarez, Verdu point out the background issue. [Citation]. Quickenden et al. (2004) recommend that if a surface is suspected to be interfering with the luminol reaction, a part of that surface which is less likely to be exposed to blood, such as a part of the surface which is covered with an object, should be tested for background chemiluminescence. Thus luminol also has background issue.
Therefore, before spraying the Hemascein reagent(s), use your alternate light source on the target area and take pictures as a negative control to know of any potential background fluorescence that you may need to account for later. Continue taking pictures after you begin spraying every few seconds.You may continue photographing until 10 minutes as the background may potentially increase after this time depending upon type and condition of substrate.
Second, it is advisable that a portion of substrate (or similar substrate) without blood may be treated with Hemascein to observe any potential background staining prior to applying Hemascein to the entire substrate or crime scene to understand what you may expect to see under those specific conditions (this is a good idea even if you are using luminol as Quickenden et al. suggests).
Third, make sure you mix the stock solution well before use so that it is in its reduced state and not oxidized state. The zinc particles in the solution catalyze the reaction; therefore, a good mixing will ensure better contact of the fluorescein reagent with zinc particles. The vial may feel warm, therefore hold it carefully with precautions while mixing.
Fourth, mix the working solution well before each use.
Fifth, spray the Hemascein® solution and the Hydrogen Peroxide solution at least 12 inches above or to the side of the target surface and let the mists drift to the target. Do not overspray. Usually just one spray is enough unless it is a carpet where more than one spray may be required. It is important to not over spray and to give time for a reaction. [Citation]
Lastly adobe Photoshop may be used effectively to successfully eliminate any such background [Citation]
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Alternate Light Source (ALS), Goggles and Filters
I am not sure if an Alternate Light Source (ALS) would be convenient for me to carry at the crime scene. Is it very light and small?
First you carry a lot of things at crime scene. An ALS can be as small as it fits in your pocket (Catalog # ALS014LED). You rather have a reliable, safe and DNA friendly technology such as Hemascein for identifying latent bloodstain than using something that has the potential to jeopardize DNA profile, make you worried about your health due to its potentially carcinogenic nature, inconvenience of completely darkening the entire room and dealing with their short lived shelf life. Moreover ALS is used in other critical applications at crime scene. You also need to wear orange goggles. There is a training video on our site that you may watch 24 hours a day at your own convenience.
What are other uses of ALS?
ALS can be powerful at a crime scene. It may provide important data point on the scene even before spraying of Hemascein as a negative control. Luminol does not allow so as it does not require an ALS. A negative control gives you the confidence on your result.
How much does it cost?
As low as $49.00.
Why do you need to wear goggles or need to use filters when shooting with your digital camera?
Hemascein works on the property of absorbing light of short wavelength and emitting light of longer wavelength. Thus the reaction must be observed at a wavelength greater than the incident excitation light, and therefore a filter is required to screen out any reflected incident light or other competing light. To achieve this filtering effect, a series of different-colored goggles can be used. The shade of goggles worn (yellow, orange, red) becomes darker as the wavelength of incident excitation light is progressively shifted toward the longer wavelength end of the Spectrum. These colored goggles are ‘‘long-pass’’ filters that let light through above a certain wavelength while cutting out light below that wavelength.
Shall I use Deep Yellow or Orange Goggles/Filters?
Either should be fine as the wavelength of both is greater than the incident excitation light. Some crime scenes use both. Orange filters/Goggles are commonly available and thus used more frequently. Use of a specific goggle/filter type (or both) should be established when evaluating/validating the test. You can download a guide on filter selection here.
How much do orange goggles cost that I need to wear?
As low as $19.00. You can also use orange gel paper sheet instead that cost less than $2.00
What wavelength of ALS may I use?
The wavelength should be between 415-480 nm. You may use an ALS with wavelength of 455 mm as it is widely available.Do not use UV light (below 400 nm).
Is too much light bad? Is polilight not recommended for this reason?
Strong lights are not recommended as they can saturate the sample with light. Polilight is considered a very strong light and thus may be used but with caution by not focusing directly on the substrate.
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How is ABAspray different than others?
A fine and consistent mist is critical to your investigation. ABAspray produces the finest mist when compared to the most commonly used hand pump sprayers. It has a unique nozzle assembly and the mist produced is valuable in not only detecting latent blood but also in preserving the morphology and spatial position of blood stain pattern.
It is important to not over spray and to give time for a reaction. Spray the Hemascein® solution and the Hydrogen Peroxide solution at least 12 inches above or to the side of the target surface and let the mists drift to the target. Usually just one spray is enough unless it is a carpet where more than one spray may be required.[Citation]
The Figure 4.45 shows that Hemascein was successfully able to resolve bloody lines whereas luminol reagents had mostly hard time doing so on non-porous vertical surfaces.
Figure 4.45: Images showing the ability of each of the reagents to reduce blood pattern
distortion on vertical surfaces.
Which sprayer is the best?
An ideal sprayer is the one that works using compressed gas reservoir like the one for paint sold at home improvement stores like Home Depot. However it is not only expensive but heavy to carry around, contain hazardous gases, can bring contamination to the crime scene, hard to clean up, require extensive set up (spray width, airflow speed, solution flow speed), wastes reagent and their reservoirs may contain iron containing metal that can react with spray reagents. A false positive has been observed with luminol with this sprayer type where the iron impurity in the stainless steel reservoir reacted with luminol and the reservoir glowed blue. Thus although such sprayer is the best, it has disadvantages that can derail your investigation.
ABAspray gives you an optimal performance when compared to these heavy industrial sprayers. ABAspray is so light that it can be used at any nook and corner of a crime scene where it may be hard to reach, it does not require any compressed gases or cartridges or power, is easy to clean and disposable. Each of the Hemascein kit already comes with 2 of such sprayers.
As can be seen in Figure 4.43 below, the droplet size of ABAspray is significantly finer than commonly used hand pump sprayer but is comparable to industrial scale sprayers within margin of error in this experiment (margin of error is missing in this analysis on industrial scale sprayers).
Figure 4.43: Graph showing the average droplet size for each of the different sprayer types
In figure 4.46 you can see that ABAspray causes least running among others minimizing the distortion of blood stain pattern than other sprayers.
Figure 4.46: Images showing the ability of each of the different spray types to reduce blood
pattern distortion on vertical surfaces
The table below summarizes the differences between sprayers.
|
Cost |
Ease of transport |
Ease of use |
Life time |
Capacity |
Pump Sprayer |
Inexpensive |
Easy |
Easy |
Until broken |
5L |
ABA Spray |
$15 USD
(ABA) |
Easy |
Easy |
Disposable but can be used more than once |
200mL |
ABA finger |
- (ABA) |
Easy |
Easy |
Disposable but can be used more than once |
5mL |
ECO Spray |
$60 USD
(Bluestar) |
Easy |
Easy |
Gas canister needs replacing ($22 US) |
250mL |
Spray gun |
Expensive (gun+
either compressor or nitrogen
tank |
Moderate to difficult |
Moderate, but need
to find a power point if using a compressor and have to carry compressor/nitrogen tank around crime scene |
Nitrogen tank
needs replacing |
100mL |
Table 5.2: Summary of the economical and practical parameters for each spray type
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Hydrogen Peroxide Tablets
Why cannot you provide hydrogen peroxide tablets so that I do not have to buy it?
Hemascein technology is not where you mix hydrogen peroxide together with the Hemascein reagent right before spraying as you do in some tablet formulation where tablets are mixed together. Both Hemascein and Hydrogen Peroxide must be sprayed separately. Watch a training video on our site.
Still why cannot you provide tablets so I don’t have to buy hydrogen peroxide?
You can buy such tablets from sigma for as low as $0.06 per tablet. Your mixture may not last long as we believe that the stability of such tablet formulation is very short lived. Rather the liquid hydrogen peroxide (1-3%) is easily available that you can buy at most medical store. This concentration (1%-3%) is routinely used on wounds and is safe and can be stored for a long time. (Moreover there is a question on stability and transport of these tablets and MSDS mandate that they be stored at cold temperatures).
I hear that Hydrogen Peroxide tablets contain more than 65% Urea that denatures DNA?
Yes . Urea is used to stabilize these tablets.
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Why is Zinc required?
Zinc reduces fluorescein to fluorescin and it becomes colorless. Therefore, a good mixing of the stock solution ensures better contact of the fluorescein reagent with zinc particles. The vial may feel warm, therefore mix it carefully with generally accepted precautions. The brightest form of fluorescin is thought to be the dianion, which is the predominant species at about pH 9. (Thefore the aqueous Hemascein formulation is prepared in NaOH for this reason.). Finally, H2O2 reoxidizes the fluorescin-heme complex and promotes the fluorescence.
Is Zinc safe?
Zinc is NOT consisted hazardous waste by Environmental Protection Agency (EPA) and is NOT one of the 8 RCRA metals (Arsenic, Barium, Cadmium, Chromium, Lead, Mercury, Selenium, Silver). You can download the P-list and the U-list for discarded commercial chemical products that classifies the material that are hazardous and lists the ones that are Toxic or Reactive with the prefix (T) and (R). Zinc as used in Hemascein formulation is not listed on either of these.
But I have heard that spent zinc is harmful for fish?
Zinc is used in fertilizers and used in boat materials to prevent rust. If factories dispose tons of zinc in sea water, it may be regulated (like a slag from a zinc processing plant, plant that does zinc plating on carbon steel , sludges from the manufacturing of motor vehicles using a zinc phosphating process, factory manufacturing zinc yellow pigments). Thus quantity of zinc matters like a dose of a prescription drug matters. Ultimately you have a choice and you can chose to dispose it like you dispose other crime scene waste. Zinc is not harmful in a vial of Hemascein for fish, due to the miniscule amount.
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How does ABAfix work?
ABAfix is used as 0.5 gram per 100 ml of Hemascein formulation when spraying on vertical non porous substrates so that running is minimized. However, one must be careful in not clogging sprayers and its must be tested before use. It can be purchased separately (based on availability) but not supplied with kit as ABAfix is not required in every situation
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Is Hemascein potentially Carcinogenic like luminol is?
No, the dye used in Hemascein is also used in eyes by ophthalmologists for decades.
Luminol is a suspected carcinogen, its health risks are unknown. Moreover, people who have to live in a house or a car after the house/car has been processed with luminol reagents may be at a risk and it is the law enforcement's responsibility on its clean up as per Department of Justice.
Did you know that significant liability claims have been paid to clean up a car or a house once sprayed with Luminol containing reagents. Read here in the study (Slide 29) .[Citation][Citation]
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I hear it is quite easy to work with Hemascein?
Hemascein is fairly easy to use when compared to a crime scene personnel preparing to study a crime scene. You simply need to prepare Hemascein solution that takes less than 10 minutes, It is quite stable that you do not need to prepare at the crime scene but can premix it before coming to scene. Hydrogen Peroxide solution does not require any preparation. In addition you will need an ALS (small and light weight that would fit in your pocket). Watch the training video at your own convenience.
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Photography Camera Settings
What camera setting may I use?
You must confirm the ability to take good pictures with your specific camera, light and filters before using at an actual crime scene.
Exposure time: 1/2 to 30 seconds
Aperture: f / 9
ISO equivalent: ISO 200
Focal length: ~50mm
Format: JPEG or RAW (both recommended if doing a comparison such as footprint or a fingerprint)
For best results, use a tripod and do not use flash
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Cleaning of Hemascein at a Crime Scene
How is cleaning of Hemascein different than Luminol & Luminol based tablet reagents?
Hemascein formulation spray components are non toxic. Hydrogen Peroxide (1-3%) solution is used as an antiseptic. Therefore, if you require to clean the surface on which Hemascein was sprayed as per the protocol, you can simply wash it with plenty of water. You can also use soap and regular cleaners.
"We spray lightly and give time to react. We have never cleaned up after an application. We have often when we used luminol but not with Hemascein." (Personal Communication with a Crime Scene Professional).
Luminol based formulation have been linked to carcinogenicity/toxicity and mandates wearing of respirators. Did you know that significant liability claims have been paid to clean up a car or a house once sprayed with Luminol containing reagents. Read here in the study (Slide 29) .
Hemascein: Discovery and Testing of Human Blood
Validation of a novel latent bloodstain detection kit
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It can be requested here.
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I have additional questions?
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