Forensic Serology: Quiz 2

Leucomalachite Green (LMG)

A reagent used in a presumptive test for blood. *This is what we use.* - positive = green - not as sensitive as TMB or specific as phenolphthalein. - used for screening tests ONLY - quick and easy to perform Sensitivity... - 1:10^5 dilution of whole blood Specificity... - specific against other biological fluids, plant extracts, and inorganic compounds

Tetramethylbenzidine (TMB)

A reagent used in a presumptive test for blood. A component of Hemastix, a kit for field tests. - positive = green/blue green - a plastic strip with filter paper on tip containing TMB and peroxide. - Cotton swab is moistened with water, blood stain is sampled. Blood smeared swab is rubbed on Hemastix.

Antigen

A substance that will cause the production of specific antibodies when introduced, or will react with antibodies in a visible way. in a blood sample, the serum protein is this.

Hematin chloride

A type of Takayama test (?). A microcrystal test. - chloride salts under acid conditions react with hemoglobin to form this. - forms brown rhomboid crystals.

Hemochromogen Test

A type of Takayama test (?). A microcrystal test. Sensitivity... - 1ul of blood (0.1mg hemoglobin), 5ul of 1:1000 dilution of whole blood. Specificity... - false positives with heme containing enzymes catalase and peroxidase.

Hemoglobin

An iron-containing protein in red blood cells that reversibly binds oxygen. - 2 alpha polypeptides - 2 beta polypeptides - 1 divalent heme unit per polypeptide chain

Ouchterlony Double Diffusion

Blood identification technique. This test methodology involves cutting three wells into a blank gel medium and adding known antibodies to one well, known antigens to another, and patient sample to the third. - antiserum placed in center - several blood stains tested at one time - white line means antiserum and blood interact - cross reactivity between similar species can occur - titer must be balanced between Ag and antibody. Shape of the precipitin bond may vary due to imbalances in titer.

Ring Precipitin Test

Blood identifying technique. An immunoassay test in which a soluble antigen reacts to form a precipitate when it combines with a specific antibody in the presence of an electrolyte. - blood sample (dilute) in top layer - precipitate means blood and antiserum species match - antiserum in heavy bottom layer

Fluorescein

Combined with an oxidant and sprayed over area thought to contain blood. - fluoresces when treated with a UV light - includes a thickener, and is more effective on vertical surfaces. - studies show no interference with DNA analysis

white blood cells (leukocytes)

Contain DNA in the nucleus. A type of blood cell. - neutrophils, eosinophils, basophils, monocytes, B&T lymphocytes.

red blood cells (erythrocytes)

Contain no DNA. A type of blood cell. - contain the protein hemoglobin (carries oxygen)

Confirmatory tests

Due to the possibility of false positives with presumptive tests, these are necessary. - involve making crystals that detect the presence of hemoglobin. - species testing and DNA testing may also be considered one of these tests.

antibody

In a blood sample, this is produced when foreign serum protein is detected. - an antiserum - certain ones will only attach to one species' serum protein, which is how a species is identified. Molecules found in the serum (immunoglobulins) formed in response to an antigen. Synthesized by lymphocytes found in the spleen, thymus and bone marrow.

aging and environmental factors.

Microscopic examination is greatly affected by...

High dose hook effect

Occurs in the ABAcard HemaTrace test. This effect occurs when hHb concentration is so high that free hHb blocks the formation of the Antibody-Antigen-Antibody complex, resulting in no dye formation in the test area. hHb antibody-dye conjugate in excess of hemoglobin cannot bind to the antibody in the test area, but can bind to an immobilized anti-immunoglobulin in the control area.

Heme tests

Presumptive test. - most are based on the "peroxidase-like" activity of hemoglobin. - catalyzes the oxidation by peroxide of organic compounds to yield colored products. AH2 + ROOH --hemoglobin--> A + ROH + H2O

positive

Rapid color change in a presumptive color test is a ____________ result, meaning the stain COULD be blood.

plasma

The blood is about 55% __________ and 45% cells. Also contains platelets. All blood cells have blood groups on the outside of their cells.

Chemiluminescence and Fluorescence tests

Types of presumptive tests. These are more sensitive than color tests, but may damage the blood stain. - cannot distinguish blood or DNA from one person to another. - used to locate and define areas of blood (old bloodstain, cleaned flood).

Porphyrins

Used to identify blood. a group of light-sensitive, pigmented, ringed chemical structures that are required for the synthesis of hemoglobin. - derived from the cyclic ring compound porphin - has the ability to combine with metals (Fe, Mg) - heme is ferroprotoporphyrin Fe2+ = ferro, Fe 3+ = ferri

- benzidine - phenolphthalein - O-tolidine - tetramethylbenzidine (TMB) - leucomalachite green (LMG)

What are five types of presumptive color tests?

Luminol: requires almost complete darkness, diminished luminescence during second application, lab prep is needed, no shelf life after mixing, not destructive to DNA. Bluestar Forensic: doesn't need complete darkness, maintains same luminescence during second application, easy to mix in field, can be used several days after mixing, not destructive to DNA.

What are some differences between Blue Star Forensic and luminol?

- stain too old - stain too dilute - substances may interfere with reaction

What are some reasons why a stain might not react in a presumptive color test?

1. visual examination of evidence 2. presumptive screening test (is it blood?) 3. confirmation test (seriously, is it blood?) 4. determine species origin (is it human?) 5. Identify the blood (whose blood is it?)

What are the steps for analysis of blood generally? What are the questions you should be asking?

- sample stain with clean cotton swab, or cut out part of the stain and put it in a spot plate - apply chromogen - apply oxidizing agent (H2O2) - The catalyst of the reaction is hemoglobin

What are the steps to performing a color presumptive test?

chemical oxidants, plant materials

What other non-blood substances could catalyze the reaction in a presumptive color test besides blood?

Phenolphthalein

a reagent that is used in a presumptive test for blood. - positive = pink - some other substances (other than blood) can produce colors other than pink. - more specific, and not affected by vegetable peroxidases.

Fluorescence

light is emitted when a substance is exposed to a shorter wavelength of light

Platelets (thrombocytes)

one of the formed elements in the blood that is responsible for aiding in the clotting process. - liquid component is plasma - serum

Chemiluminescence

process by which light is emitted as a product of a chemical reaction

Presumptive tests

produce a color reaction or release of light. Tests rely on catalytic properties of blood (hemoglobin presence). Reaction participants include... - chromogen (*color changing chemical*, i.e. LMG, TMB, Luminol, etc.) - H2O2 (*oxidant*) - Catalyst (hemoglobin or peroxidase) Donor + H2O2 <--> Oxidized Donor + H2O

DNA typing

the means of distinguishing the DNA of one person from another

Luminol

the most sensitive chemical test that is capable of presumptively detecting bloodstains diluted to as little as 1 in 100,000; its reaction with blood emits light and thus requires the result to be observed in a darkened area. - combined with oxidant and sprayed over area thought to contain blood. - mechanism is greatly unknown - hemoglobin greatly enhances chemiluminescence upon oxidation in alkaline solutions. - the hemoglobin derivative hematin is the actual catalyst, therefore, this often works better on old stains. - produces max luminescence at 441nm with a shift to 452nm with older stains. Sensitivity... - from 1:5x10^6 to 1:10^7 dilution of whole blood. Specificity... - false positives: cupric salts, copper alloys, ferricyanide, hypochlorite, iron chlorophyll. - negative for mild, coffee, other biological fluids, grass. leaves, wood, earth. - doesn't interfere with most serological tests, as well as PCR. May interfere with takayama test. Hydrogen peroxide make interfere with the reaction at high concentrations.

Species origin

The species from which an item of biological evidence originated, such as human, horse, cat, cow, etc. Most methods test for *serum proteins*. - serum proteins are found in all animals, but are slightly different. - species ID methods are based on antigen/antibody interactions. - based upon the reaction of antigens found in blood, forming an insoluble complex that precipitates in a detectable form with antisera produced against a specific antigen. Precipitation occurs due to extensive cross linking between antigen/antibody complexes. The large size of these lattices, and the masking of polar groups during complex formation causes precipitation.

Takayama Test

*Confirmatory test.* A microcrystal test for blood. A small amount of blood is added to a microscope slide. Chemical solution is added, and the slide is heated to form crystals. The crystals are then viewed under the microscope. - saturated (D)-glucose and pyridine under basic conditions react with hemoglobin to form hemochromogen. - crystals are salmon-pink rosettes. - often more reliable with older stains. - the speed of formation is *temperature and solubility dependent*. - a negative test doesn't necessarily indicate that blood is absent.

protein reacting agents

- dyes that adhere to blood proteins - useful in the visualization of imprints - Coomassie blue, amido black, rhodamine

False positives

A positive result given by a substance that is not blood.

Kastle-Meyer test

A presumptive blood test using phenolphthalein and hydrogen peroxide. If blood is present, the indicator immediately turns pink/purple. - solution is stable for 1-2 hours - phenolphthalein is reduced by refluxing with a strong base. - 2 stage test Sensitivity... - 1:10^7 dilution of whole blood - 1:10^6 dilution of old, decomposed blood Specificity... - false positives can be caused by saliva, pus, vegetable extracts, heavy metal salts, or feces of patients taking aspirin. - negative for rust, urine, semen, sweat and milk.

O-Tolidine

A reagent that was once used in a presumptive test for blood. No longer used because it can metabolize to benzidine, which is carcinogenic. It was gradually replaced by TMB. - positive = blue

Benzidine

A reagent that was once widely used in a presumptive test for blood. No longer used because it was found to be a carcinogen (causes cancer) in 1974. - positive = blue

ABAcard HemaTrace

A blood identification technique. - specific for human hemoglobin (hHb) - sensitive to 0.05μg/mL - the average concentration of hHb is 150,000μg/mL - our validation shows dilution of whole blood to 1:256,000 is still detectable. hHb reacts with mobile monoclonal antihuman Hb antibody with conjugated due particle in the sample application region. Antigen-antibody complex migrates to the test area where a polyclonal antihuman Hb antibody is immobilized, Antihuman Hb antibody captures the complex forming an antibody-antigen-antibody sandwich. Pink conjugated dye particles concentrate in the test area.