jimtrue.com : school : CJT2260 : 2003-09-04: Presumptive Tests for Blood
Posted by Jim True on September 4, 2003 6:14 AM. Last Updated October 22, 2006 9:23 PM
Disclaimer for all material noted here is at the bottom of this web page.
Presumptive is for giving possibility of blood present.
Concentrating tonight on Kastele - Meyer (Phenolphthalein) and Luminol
Definition of "Presumptive"
Presume: to take for granted; assume to be true until disproved, to indicate the probability of or to seem to prove.
Presumptive: Creating or affording reasonable grounds for belief; based upon presumption. Only will give an indication, not an affirmative.
Meaning:
The results of a presumptive test will not conclusively prove or disprove the presence of blood.
In testimony, "The results of the test indicated that there may have been blood present," or, "there was no indication given from the test results that blood was present."
Hallmarks of Good Presumptives
* Sensitive and specific to heme. (Iron portion of our blood)
* Relatively stable and non-toxic. (Won't want to use if it will cause brain cancer, or if you have to make up a fresh batch everytime you go out to use it).
* Reliable and economically feasible to use. (Know what to expect and can't cost $200/oz to makeup)
* Non-destructive to evidence. (First test should NOT be your last)
* Sensitivity allows for indirect testing. (Less material that is tested or destroyed in the field, the more material to be used in the lab - or take sample onto a swab).
* Meets the Frye standard for general acceptance. (Everyone in the scientific community that this test is sound, reliable and accepted).
Hemoglobin
* Responsible for oxygen and carbon dioxide transport.
* Contained in red blood cells (erythrocytes). The iron imparts a red color to blood (Ferric). Horseshoe crabs have blue blood because their blood is copper based.
* Hemoglobin has four protein chains (2 alphas & 2 betas), with the iron atom nestled inside.
* Globin = protein component, heme = iron component.
Hemoglobin & Peroxidase-like Activity
* Peroxides are toxic to animal tissue (Hemoglobin breaks down peroxides, that are toxic to our bodies).
* Peroxidase (HRP) is a hemoprotein which catalyzes the oxidation of hydrogen peroxide (breaks down the hydrogen peroxide).
* Peroxidases break peroxides apart into water and free oxygen radicals, rendering them less harmful. [Important later for understanding how Kastle-Meyer works]
Animal & Plant Peroxidases
* Animal: (Myeloperoxidase-like, found in humans and dogs; prostaglandin H2 synthase, found in sheep and mice)
* Plant: (cytochrome-C peroxidase-like family; peanut, horseradish, yeast, barley and various fungi.) Also turnips, potatoes, etc. Sometimes also cucumber. None when crushed up are really going to look like blood.
Animal & Plant Peroxidases (very important Differences)
* Animal peroxidases can withstand the prolonged heat of an arson scene
* Plant peroxidases are relatively weak and give slower reactions (if any) to presumptive tests. Visual exam will usually clue one in if encountered at crime scenes.
A Visual exam performed at the outset will clue you into the presence of blood; as opposed to a Polish/Italian wedding gone horribly wrong.
The Visual "Exam"
* An evaluation made by looking at a stain and determining its probable nature.
* Color, consistency, texture, etc. (Sometimes also the odor).
* Conserves time and chemical use.
Sensitivity and Specificity (Important!)
* Sensitivity: The amount by which blood may be diluted and still give a reaction to the test. (ie: 1:10,000 ml in KM; Luminol will pick up blood of 1 part to 1,000,000 mL of water).
* Specificity: The likelihood that a test will give a true positive reaction ONLY when exposed to blood. (High to low). Specific to blood and no other matter. KM reacts with almost no other substances other than blood; Luminol reacts with bleach and several other materials as positive.
Interpretation of Results
* Positive: color forms within a certain time frame after all reagents are applied. ie KM, you would expect pink color change only AFTER Hydrogen Peroxide was added, not before.
* Negative: No color formation is observed after application of all reagents.
* False positive ("bad test"): color forms after SOME reagents are applied. Result of interference. No determination can be made regarding blood's presence. _Bad Test is better terminology for False positives._
* False negative: no color forms. May be no actual blood present, or the blood is too dilute for the test. May be interference (ie ascorbic acid, Vitamin C).
Tetramethylbenzidene (TMB)
* Gives a green color in presence of blood.
* Sensitive & specific to heme.
* Causes bladder cancer with prolonged use / exposure.
* Best used for development of blood prints (especially on reddish substrates).
Kastle-Meyer
* Forms a pink color in presence of blood. (hot flourescent kind of pink)
* Sensitivity 1:10,000.
* Highly specific (indicative) for blood.
* Uses relatively non-toxic reagents.
Luminol
* Reaction produces light instead of color. (Luminesence, not phosphoresence, or flourescence, or effervesence).
* Sensitivity 1:1,000,000.
* Specificity is low due to Luminol's propensity to react with many substances other than blood. (The 'ho' of presumptive tests, likes cleaning agents, chrome, nickel, nickel agents, bleach, oxidants).
* Good for examining large areas, especially where clean-ups suspected.
* Luminol could continue to luminesce for up to 3-5 minutes.
* Unlike TMB and KM, Luminol is a direct test. You will spray it directly onto evidential area. KM or TMB you will take a sample and test that sample.
* Will not interfere with subsequent DNA tests.
* Should _never_ be used on visible bloodstains. Good for occult areas (occult meaning 'hidden').
* "Back up" results with a KM test (since KM is more specific). Swab the area you see luminescing and then check it with KM.
Typical KM (phenolthalein) test kit
Swabs, Alcohol, Pheonolthalein reagent, Hydrogen Peroxide. (Distilled water), and a Known Bloodstain control.
Using Kastle-Meyer (Controls)
* Positive control: swatch with bloodstain serves as a known sample for testing reagents.
* Negative control: use any area reasonably expected to be free of blood. Not included with the test kit; this is taken at the scene or some other area that you can be reasonably certain there is no blood. There should be no reaction; if you get a result from the negative control, it means your reagents are probably contaminated.
* If reactions are reasonable, proceed.
Using Kastle-Meyer (Transfer materials)
* Transfer materials allow one to test part of the stain without compromising the evidence itself. Should be 100% cotton.
* Swabs (cotton)
* Filter paper
* String / thread (cotton)
Using Kastle-Meyer (Reagents)
* Alcohol: increases sensitivity by "cleaning up" debris (background clutter). Expose the heme and make the test more sensitive that way.
* KM (reduced "oxygen-hungry" _phenolthalein_): color indicator. Turns pink when oxidized. (Prepared by taking 20 grams of pheonolthalein, zinc metal dust, sodium hydroxide, di water, and boiled in a round bottom flask, which will drive the oxygen out of the solution and will turn clear).
* Hydrogen peroxide (H2O2): Releases an oxygen radical in presence of heme. THIS oxidizes the KM.
Three steps in the KM test; three reagents.
* De-ionized (D.I.) water: not a reagent, per se, but may make transferring material to the swab easier.
* Be careful not to use too much -- you'll over-dilute your sample! Could give a false negative reaction.
Using Kastle-Meyer (Process)
* Positive / Negative controls; run these first.
* Swab periphery of stain (save good stuff for the lab!) Only use the edge; save the middle and primary portion of the evidence for the lab.
* Do not apply reagents to swab while holding it over the evidence, please! You could spill and contaminate the evidence.
* Apply alcohol to swab. One drop is sufficient.
* Apply KM (phenolphthalein reagent). One drop is sufficient.
* WAIT for a few seconds to see if a pink color forms. If it does, stop the test.
* If no pink color forms, apply hydrogen peroxide. Color change should occur almost immediately.
* Do not "read" results after 30 secs. It will change due to oxygen from the ambient environment and will oxidize the color change.
How does the KM test work?
* Hemoglobin has peroxidase-like activity.
* It breaks down H2O2 into H2O and O.
* The "O" oxidizes the KM. Therefore..
* Hemoglobin will cause hydrogen peroxide to oxidize KM and turn it pink.
Tracy's website link for KM process.
Luminol (3-aminophthalhydrazide)
* Developed by Dr. Walter Specht (Jena, Germany) in 1937. Initially used to help miners find copper deposits.
* Produces luminescent reaction (light) (451 nm) instead of color.
* Best viewed in total darkness due to weak luminescence.
Luminol
* Sensitivity: 1:1,000,000
* Specificity: low.
* Reacts with blood, and also...
* Chemical oxidants (bleach, hydrogen peroxide, some detergents, etc.)
* Chemical catalysts (something that doesn't get changed itself) (Copper, brass, nickel salts, chrome fixtures, etc.)
* Plant peroxidase (based on Extreme sensitivity, mostly.)
* Mosquitoes and lovebugs (hit and run on these bugs that have sucked blood).
Low Specificity = Back Up Your Results!!
* Since Luminol reacts with so many things, use KM (higher specificity) to test the area which lumnol "tells" you that it contains blood.
* "Twinkling" reaction typical of catalysts (metals). Intense, but short-lived.
Luminol reagent components
* Luminol: takes place of color indicator.
* Sodium Carbonate (Na2CO3): Adjusts pH to make the solution basic (slippery, or soapy).
* Sodium Perborate (NaBO3): Oxidant. Heme breaks this up to oxidize the luminol.
Application
* Reagents are mixed with DI water. Should be used within 8 hours, optimally.
* Get familiar with surroundings before cutting off lights!
* Use positive control before beginning test. Pennies are good for positive controls and for marking distance.
* Spray walls first (spatter patterns), followed by ceiling (cast off patterns). Do the floors last. Shoe prints, drag marks, etc.
Documentation
* It is best to apply spray only ONCE. Subsequent applications will further dilute any blood present. (Blood is hydrophilic.)
* Blood prints MAY run.
* Have photo/video equipment set up and ready along with luminescent rulers, etc.
* Time exposures, B&W film.
* "Light flash" method for room.
Anytime you get a postive reaction with Luminol, ALWAYS ALWAYS ALWAYS back up the test with KM.
Disclaimer: These are MY notes taken from classroom lectures while I'm in the classroom. While I'm perfectly happy to share my notes with my classmates and I know I take very good notes, you should still make every effort to attend the class and TAKE YOUR OWN NOTES. I will not transcribe everything the instructor says in the classroom, and I will NEVER post pre-exam reviews. My notes will not replace the value of actually attending class and taking your own class notes.I also cannot attest to their accuracy, other than they are what was provided in the lecture; you should not reference my notes as "expert opionion" by any means, and if you notice an error or omission, please do me the favor of e-mailing me with the correction and I will re-post my notes. End of Disclaimer.