Physical Evidence: Blood and Blood Stains

Representative Objects and Materials Constituting Blood Evidence

Blood stains (and their associated bloodstain pattern-types) fall into general categories, with blood stains comprising the actual “drops deposited due to the effects of forces applied to the blood itself, including impact and gravity. Bloodstain patterns branch out from these in association with how the stain was made. In categorizing blood stains, the stains themselves (with their associated patterns) end up making up the bulk of the collectible and examinable evidence. This includes both liquid and dried stains, no matter what substrate they are deposited on. Blood is a liquid and, as such, is subject to the rules of liquids prior to its drying; after that point, it would be subject to any sort of flaking or powdering rules (meaning that, yes, flakes, powders, and dried stains also comprise this type of evidence).

To put it simply, there are three basic groups into which these can fall: passive stains, transfer stains, and impact stains. Transfer stains include voids, bloody footprints, wiped stains, and so on (any stain transferred from one surface to another); passive stains are, more or less, those most created by the effects of gravity, including drip stains or tails and any pooling, saturation, and combinations of parent and satellite stains; and impact stains would include those stains most affected and caused by forces other than gravity, like spatter stains and resulting cast-off or cessation patterns.

Also associated with this type of evidence would be blood clots and the measurements necessary for calculations; calculations will be expanded on more in a moment, while blood clots will not (though an understanding of blood is important to understand blood evidence and how it is collected— and, indeed, how aspects of it are calculated). 

Chemical and Physical Tools and Techniques used to Analyze Blood Evidence

At the scene, when the stains are photographed, the use of a scale next to the stain and, ideally, a secondary scale in addition to it for the sake of gathering the width and length of the stain will allow the forensic scientist to calculate important facets of the stain’s context. The width and length are incredibly important; so too can be the position of satellite stains, the distance between drops in a drip pattern, and so on. Taking measurements then becomes incredibly important as far as physical evidence is concerned. 

In testing blood stain evidence, it’s important to remember that any forensic scientist must start from the base assumption that they are not dealing with blood when they are testing it; as such, the initial chemical testing they run on any piece of evidence received must be presumptive. Presumptive tests for blood include benzidine, tetramethylbenzidine, leucomalachite green, phenolphthalein, o-tolidine, and luminol, each of which is catalytic and requires a peroxidase (like hydrogen peroxide). In short, a presumptive test is used, utilizing a reagent and a peroxide to catalyze the reaction (which will be expanded on later). If there is a positive indication for blood based on the relevant mechanism of the chosen presumptive test, a confirmatory test can occur. This requires the reagents (the indicator, oxidant, and so on) and some sort of swabbing agent. 

An examiner can also use, for the sake of presumptive testing, something like Hemastix, which will operate on the same color change principles and presumptive techniques. 

Forensically Valuable Properties of Blood Evidence and the Chemical and Physical Processes That Create Them

Physically, blood stains can be measured such that their angles of origin, convergence, and impact can be calculated. From these, crucial inferences can be extrapolated; but these must be calculated before any of that. The direction of each stain is of incredible import and should be noted; each stain’s direction can be determined based on its pattern and calculable characteristics, and direction tends to become more calculable the more elliptical it becomes due to the strain and tension of liquid blood pulling at the drop itself as it travels. Because of this, the angle of impact can be calculated by the formula θ=arcsin(width/length), where the width and length are measured from the bloodstain itself. The more elliptical the bloodstain, the more acute the angle of impact tends to be; the opposite is true for more right- or obtuse-angled bloodstains.

You can also calculate the drop height based on diameter. Generally, as the drop reaches terminal velocity, calculable based on the general rate of gravity relative to the mass of a drop of blood, diameter increases (i.e., width increases). The roughness, porousness, or polishedness of a surface has an impact on how the blood spreads (as seen with different kinds of stains and how they gain satellites or begin to seep in); the elasticity of these surfaces then has an effect on their tendency to splash and their diameter as well. As the drop splashes, it gains spines that can be used to calculate this drop height more fully. 

Additionally, there’s a tendency for some energy to be lost during the drop and a need for cast angles to be accounted for in this calculation. Despite this, the drop height of a circular bloodstain can still be generally calculated from a combination of the number of spines on the drop (small projections/disturbances out from the exterior of the ring) and  the diameter (the stain’s width) over a fairly limited range of angles of impact. For the same limited range of impact, elliptical ranges can be calculated in the same way. 

Velocity of spatter is calculable by diameter by the same ideas; low velocity spatter tends to be larger than medium-velocity spatter, whereas high-velocity spatter, like mist spatter in the classic impact stain category, would be smaller than 1-millimeter in diameter and appear like the production of a fine mist-like spray. Each of these is produced by impact from a different type of object and production of force (i.e., velocity of impact). Low-velocity impact would be produced by minimal force, as from gravity itself; think of a fall, someone’s foot, or a fist— or, perhaps, from blood dripping. These tend to be separate or compounded, with much larger diameters in the range of four to eight millimeters. Medium-velocity impact spatter, in contrast, is in the general range of one to four millimeters and can be caused by impacts that are higher-energy than gravity but less than, say, a gunshot. High-velocity impact spatter, then, would exceed that; think of a wound caused by, yes, a gunshot, but also by machinery and even the flow of blood caused high-pressure arterial spray. It’s that typical mist of high-pressure bloodflow and those tiny drops typical to it. 

Beyond the height and angle of impact, there are other calculable properties of blood stains that one can gain from those same properties that should be recorded at the scene through studious documentation. The area of convergence is found by taking the longitudinal axis of several individual bloodstains in an area and finding the point where they meet on an imagined two-dimensional plane. By taking this point, putting it into three-dimensional space, and using the angle of impact for each bloodstain relative to the area of convergence’s central pole between them, you can find the area of origin— the place that the blood very likely originated from. There is a possible error in this, of course, but it’s a good starting point. 

Chemically, there are also methods for testing for blood. It’s important to remember that each forensic investigator, when looking at a piece of evidence, is supposed to start from the non-biased position of this is not blood. As such, each test performed is presumptive. 

Though benzidine has some probative value, it is a known carcinogen; the same is likely true for o-tolidine and tetramethylbenzidine. It’s for this reason that, when available, leucomalachite green, phenolphthalein, and luminol are preferred. Phenolphthalein, which has very wide applicative purposes in chemistry, is fairly easy to get your hands on, though. It’s perhaps most often used as an acid-base indicator that turns a shade of magenta when a base is present. Because blood is basic, it can indicate the presence of blood— or, importantly, any other basic material, hence its use as a presumptive test, rather than a confirmatory one. 

That is something to keep in mind with all of these chemical testing methods and, potentially, with the application of combinatorial ultraviolet-infrared photography on bloodstain evidence. Because they are non-specific to blood, they are not as accurate as confirmatory tests; and because of how they are implemented, they can be destructive to DNA evidence. When possible, DNA collection should be done long before any presumptive blood testing is done. Photography, obviously, should be done first; but it’s a good idea to make sure that DNA evidence has been collected before you begin testing for blood on any item of evidence. Additionally, when applying a presumptive blood testing method, you must make sure not to apply it to the whole of the item of evidence for the same reason— especially if using one that requires multiple tests (you want ample blood supply for both DNA and presumptive/confirmatory tests), and especially if you are going to apply hydrogen peroxide to react with the catalase enzymes within blood. 

Phenolphthalein is an important component in the Kastle-Meyer test. Here, a blood sample is treated with a combination of ethanol and phenolphthalein. If, upon the addition of drops of both and drops of hydrogen peroxide (again, to react with the catalase enzymes within blood), this is considered a presumptive positive for blood. This indicator will turn a pink or magenta color in the presence of blood, consistent with phenolphthalein’s indicator properties. 

Luminol, or 3-aminophthalhydrazide, which is still used often enough despite its potentially-carcinogenic properties, operates on the same kind of principle. (The same is true of the name-brand product BlueStar. Except for certain instances of linoleum testing, these perform the same, generally.) It is alkaline and contains hydrogen peroxide as an oxidizing agent, much like its counterparts, making it useful for the same reasons. This is much less destructive to DNA evidence than other forms of presumptive testing, which is probably why it’s used very often in scene work— that and its chemiluminescent properties. These properties make it very useful for detection and photography. When exposed to the hemoglobin in blood, luminol’s oxidizing agent reacts with the ferric heme groups in blood, catalyzing oxidation of luminol. This causes the same signature chemiluminescence at around 425-nanometers (in terms of wavelengths of light), caused by an intermediate in the reaction mechanism. Because it is a presumptive test for the presence of blood and not a confirmatory test, it is valuable for the presence of it; but it also has incredible photographic importance for in-scene work.

Beyond these two examples of presumptive testing, other presumptive tests operate on the same principles of oxidation and subsequent indication— something reacts and changes color. Each of them must also be calibrated via a set of knowns to weed out false positives and false negatives, potentially using water and blood or plant peroxidases and chemical oxidants, just to make sure the chemicals are working as designed. In the presence of blood or some other oxidant, benzidine (which, again, must be used with caution because it is a known carcinogen) will turn a deep blue within ten seconds if reacting positively; the same is true of ortho-tolidine; leucomalachite green (a personal favorite) will turn blue-green if reacting positive, due to the conversion of the compound to malachite green (oxidized) via carbon double-bonding; and Hemastix will turn the same green-ish color along the testing strip, if used. 

Following presumptive testing, a confirmatory test like a Takayama test (wherein pink, rhomboid hemochromogen crystals form after application of a specific solution), RSID test (utilizing antibodies to detect Glycophorin A), Hematrace testing, or so on, can be used. 

The Probative Value of Blood Evidence and the Range of Conclusions

The extrapolation of data from the reconstructed and calculated angles of impact and areas of convergence and origin can be very useful in reconstructing the scene itself, such that you can figure out how a scene could have gone down. Used in tandem with other information from the scene, including (but not limited to) any photography, scene scans, or any collected information on how bullets may have traveled (of used), or any injury documentation on living victims, living suspects, or decedents, you can gain a more accurate depiction of the scene for the sake of investigation.

In terms of conclusions, presumptive testing does not make conclusions on whether or not blood is present; it makes conclusions on whether or not there was potentially blood present. The actual presence of blood would be the conclusion-wise jurisdiction of the confirmatory test; for the sake of the report, your conclusion would be about the potential presence of it. Here, if there is that positive indication, caused by oxidation, the conclusion would then be that there was a positive indication for the presence of blood— nothing more. Your notes could indicate more on your process, but your conclusion report would indicate no more than that. The language recommended by the DOJ for presumptive serological testing is “indicated,” “negative,” or “inconclusive,” where “indicated” would cover your positive reaction.

The confirmatory-testing equivalent of this would be “identification,” because there is more certainty there. The issue with presumptive testing is that, as mentioned when explaining phenolphthalein, these tests are non-specific. There are many compounds that these reagents can react with; but confirmatory tests are more specific to blood and especially human blood, meaning the examiner could and should be more confident in their results.