erology s s by m. hoffmeister & h. montgomery. identification & character of blood and...

erology

erologySS

By M. Hoffmeister & H. Montgomery

Identification & Character of Blood and Bloodstains


Forensic Science• Uses blood analysis to determine the individual

(source)• Usually deals with a variety of fluids (blood,

saliva, semen, and urine) in stain form and are often degraded/deteriorated.

Environmental controls are essential ( uncontrolled heat and humidity can destroy a lot of biological information within a stain by enhancing degradation).

Serology SerologyChapter 11 & 12Chapter 11 & 12



Serology was very prominent in the 1950s to the 1980s, when DNA analysis became possible

• It is still routinely done where DNA is analyzed.• Is done when DNA analysis is not feasible• Serology is being challenged by the reliability

and the discrimination power of DNA analysis.– Important to understand preDNA serology f testimony

in cases


Protocol for the Analysis ofBlood in Forensic SerologyProtocol for the Analysis ofBlood in Forensic Serology

• Careful visualization of evidence to locate stains/ material visibly characteristic of blood.• Application of a suitable presumptive test.• Application of a confirmatory (specific and

sensitive) test.• Determination of biological/species (animal or

human) origin.• Characterization of blood using genetic markers

or DNA.


Identification of Blood Identification of Blood

A visual observation coupled with positive chemical presumptive and confirmatory tests provide sound data to support the identification of blood.

Two types of tests are given because ----NO SINGLE TEST IS ABSOLUTELY SPECIFIC

FOR BLOOD

A visual observation coupled with positive chemical presumptive and confirmatory tests provide sound data to support the identification of blood.

Two types of tests are given because ----NO SINGLE TEST IS ABSOLUTELY SPECIFIC

FOR BLOOD


Catalytic Color TestsCatalytic Color TestsCatalytic Color Tests - employ chemical oxidation

of a chromogenic (color causing) substance by an oxidizing agent catalyzed by the presence of hemoglobin (in red blood cells).

Two substances are needed for the color change:• Oxidizing agent (often hydrogen peroxide).• The heme group acts as a (peroxide – like) catalyst

Catalytic Color Tests - employ chemical oxidation of a chromogenic (color causing) substance by an oxidizing agent catalyzed by the presence of hemoglobin (in red blood cells).

Two substances are needed for the color change:• Oxidizing agent (often hydrogen peroxide).• The heme group acts as a (peroxide – like) catalyst


Catalytic Color TestsCatalytic Color Tests


Catalytic Color TestsCatalytic Color TestsCommon method of application:• Wet cotton swab with distilled water and sample the stain• Apply the reagents to the swab.• Note immediate observations – changes will take place

quickly and may not appear the same after time has passed.

False positives can usually be attributed to:• Chemical oxidants• Plant materials (with peroxidase-like enzyme).

• Animal materials that may contain traces of blood.


Catalytic Color TestsCatalytic Color TestsAdler Test (Benzidine)• More extensively used than any other presumptive test

for blood• Normally carried out in ethanol/acetic acid solution• Results in a characteristic blue color• Was deemed a carcinogen in 1974 and has been

essentially discontinued for forensic use today


Catalytic Color TestsCatalytic Color TestsAdler Test (Benzidine)


Catalytic Color TestsCatalytic Color TestsKastle-Meyer Test (Phenolphthalein)• Commonly used today• Uses phenolphthalein (acid/base indicator)• Results in hot pink color• Phenolphthalein (alkaline solution) becomes oxidized to

phenolphthalein (pink in alkaline environment)• Phenolphthalein over zinc in KOH solution• False positives do not usually produce the pink color


Catalytic Color TestsCatalytic Color TestsKastle-Meyer Test (Phenolphthalein)


Catalytic Color TestsCatalytic Color TestsO-Tolidine (ortho-tolidine)• Derivative of benzidine• Conducted under acidic conditions• Creates a blue color (similar to benzidine)• Reported as carcinogenic in rats in 1974, leading to its

replacement by TMB


Catalytic Color TestsCatalytic Color TestsO-Tolidine (ortho-tolidine)


Catalytic Color TestsCatalytic Color TestsTetramethylbenzidine (TMB)• Derivative of benzidine• Used in an acidic medium (acetic acid)• Resulting color change from green to blue/green• Hemastix (field test for blood) uses TMB and only

requires distilled water and the questioned sample (yellow to blue/green)

• Swabs with cometic materials may produce false positives


Catalytic Color TestsCatalytic Color TestsTetramethylbenzidine (TMB)


Catalytic Color TestsCatalytic Color TestsLeucomalachite Green (LMG)• Created in 1904 using the reduced form of the malachite

green dye.• Produces a green color• Carried out in an acidic medium with hydrogen peroxide

as the oxidizer


Catalytic Color TestsCatalytic Color TestsTests using Chemiluminescence & Fluorescence• The observed results often enables one to determine the

limits, shape and details of the original bloodstained area (and patterns if present)

• By nature, they are potential sources of contamination of the blood

• If the blood can be seen and collected, these probably should not be used

• Luminol and Fluorescein are irritants but not carcinogens• Chemiluminescence is the process by which light is

emitted as product of a chemical reaction• Fluorescence occurs when a chemical substance is

exposed to a particular wavelength of light (usually a short wave like, UV light) and light energy is emitted at longer wavelengths


Catalytic Color TestsCatalytic Color TestsTests using Chemiluminescence


Catalytic Color TestsCatalytic Color TestsTests using Fluorescence


Catalytic Color TestsCatalytic Color TestsLuminol (3-aminophthalhydrazide)• Luminesces after oxidation in acid or alkaline (aqueous)

solution.• The catalytic activity of the heme group accelerates the

oxidation of the luminol• Produces a blue-white to yellow-green light if blood is

present • Outlines & details are often visible up to 30 second before

additional spraying is required • Doesn’t affect these tests: presumptive, confirmatory,

species origin, ABO tests, RFLP and PCR tests• Interferes with several enzyme and protein genetic marker

systems• Sensitivity: detects dilutions up to 1 in 10,000,000• Results should be photographed b/c transient (temporary)• False positive with bleach (flash)


Catalytic Color TestsCatalytic Color TestsLuminol (3-aminophthalhydrazide)



Fluorescein• Prepared like phenolphthalein—reduced in an alkaline

(basic) solution over zinc to fluorescin.• Fluorescin is applied to the sample and the heme group

catalyzes the oxidation by hydrogen peroxide to make fluorescein.

• When treated with UV light, it will fluoresce• Includes a commercial thickener which causes it to

adhere to the surface (good for vertical surfaces).• Does not fluoresce with bleach• Shows no interference with STR testing of blood for

DNA.



Fluorescein


Confirmatory Test for BloodConfirmatory Test for Blood

Teichmann Test• First described in 1853• Heating dried blood in the presence of glacial acetic acid

and a halide salt (usually chloride) to form a hematin derivative.

(Crystal tests are often used)(Crystal tests are often used)



Teichmann Test(Crystal tests are often used)(Crystal tests are often used)



Takayama Test• First used in 1912• Involves heating dried blood in the presence of pyridine,

a glucose (reducing agent), and sodium hydroxide (basic) solution to form a pyridine derivative.




Takayama Test(Crystal tests are often used)(Crystal tests are often used)



Teichmann Test• First described in 1853• Heating dried blood in the presence of glacial acetic acid

and a halide salt (usually chloride) to form a hematin derivative.

Takayama Test• First used in 1912• Involves heating dried blood in the presence of pyridine,

a glucose (reducing agent), and sodium hydroxide (basic) solution to form a pyridine derivative.



Species Determination in BloodstainsSpecies Determination in Bloodstains

• Microscopy uses a visual comparison of the blood cell morphology (shape/appearance)



• Microscopy uses a visual comparison of the blood cell morphology (shape/appearance)

• Protein analysis (3 different techniques are used.)– Immunoprecipitation reactions (most use)

• If an animal is injected with a human serum protein, the animal’s immune system will recognize it as foreign and release produce antibodies against the antigen.

• When the antibodies and antigen come in contact and usually a precipitation is formed (precipitin reaction)



Immunoprecipitation reactions



• Protein analysis (continued)– Electrophoresis—a technique in which charged

molecules (i.e. proteins) are caused to migrate in an electric field in a suitable support medium under controlled conditions of temp, pH, voltage, and time.• Support media: starch gels, agarose gels,

polyacrylamide gels• Positively charged molecules migrate toward the

cathode (- electrode)& negatively charged molecules migrate toward the anode (+ electrode).

• Bands are visualized by a stain or chemical rxns.• In FS, it is usually done in a gel on a glass plate

w/ samples (i.e. blood soaked thread or stain extracts) are placed in the gel.



Electrophoresis



• Protein analysis (continued)– Isoelectric Focusing (IEF) - an electrophoretic

method that takes advantage of the fact that at a certain pH, a protein in aqueous solution will exhibit a point of no net charge (isoelectric point - IEP). • The buffer in the gel controls the pH throughout

the system• A pH gradient is set up in the gel w/ the low end

at the anode.• When current is applied, the proteins migrate

where they encounter their IEPs and form a band• The bands here can sometimes be more easily

observed that with electrophoresis.



Isoelectric Focusing (IEF)



• Serum Protein Analysis - serum proteins are a large collections of proteins in the serum– Ring Precipitin Test

• 2 liquids placed in a test tube: antiserum and extract of questioned bloodstain

• As the anti-human serum comes in contact with human blood sample via diffusion, a small layer of a precipitate will form between these two liquids.

• No reaction will occur if the blood is animal and used with anti-human serum.



Ring Precipitin Test



• Serum Protein Analysis - serum proteins are a large collections of proteins in the serum– Ring Precipitin Test

• 2 liquids placed in a test tube: antiserum and extract of questioned bloodstain

• As the anti-human serum comes in contact with human blood sample via diffusion, a small layer of a precipitate will form between these two liquids.

• No reaction will occur if the blood is animal and used with anti-human serum.

– Outerlony Double Diffusion Test• Carried out in a gel on a glass plate or petri dish• Wells are punched into agar, the antiserum is placed in

the center well and the extracts are in the surrounding wells.

• Immunoprecipitate lines form between the wells to show a positive response.



Outerlony Double Diffusion Test



• Serum Protein Analysis (continued)– Crossed-Over Electrophoresis

• Rows of opposing wells are cut in an agarose gel plate.• Once current is applied, the extract proteins (antigen)

move toward the anode.• Antibodies move via electroendosmosis—cations and

water of hydration move toward the cathode.• When the antibodies meet with the extract protein

(antigen) precipitin bands form between the two rows of wells

• Non-Serum Analysis includes using antihuman Hemoglobin– Hemoglobin is a protein in blood that is species-

specific for immunological properties.


Genetic Markers in BloodGenetic Markers in Blood

Blood group - a group of antigens produced by allelic genes at a single locus and inherited independently of other genes.)



Antigen-based Markers and Protein Markers• Antigen-Based Markers: Blood Groups

– ABO system• Discovered by Landsteiner in 1900• Types (A, B, AB, O) refer to antigens on the surface of

the RBC• Antibodies for the antigens (anti-A, anti-B) are present

in the plasma.• Agglutination would occur between anti-A serum and

types A and AB blood• Most individuals (80%) secrete their ABO

characteristics in other body fluids (they are called secretors).




– ABO system



Blood Types




– ABO system– The Lewis (Le) System

• Le antigens are absorbed from the plasma onto the surface of RBCs.

• Provide secretor status (whether the ABO type can be determined from bodily fluids).

– Rhesus (Rh) system (+ or -)• Discovered by Landsteiner and Wiener (1940)• Antigens are on the surface of the RBC membrane• Not present in non-blood body fluids• Natural Rh antibodies are not common in serum



Antigen-based Markers and Protein Markers• Protein Markers: Blood Groups

– Hemoglobin (Hb)• Major protein in RBCs• Phenotypes can be identified w/ electrophoresis or IEF

– Haptoglobin (Hp)– Enzyme Markers

• uses electrophoresis and IEF (Isoelectric Focusing)• Phosphoglucomutase (PGM)

– Most well known enzyme– Found in many tissues of plants, animals, &

microorganisms– In humans, it exists in significant concentrations in blood,

semen & in small amounts in vaginal secretions & cervical mucus.

– Can place within 10 subtype population groups (most discriminatory of all enzyme systems used in Forensic Science)




HaptoglobinHaptoglobin IEF – Isoelectric FocusingIEF – Isoelectric Focusing

Biological FluidsBiological Fluids

Semen • Produced by males after puberty• Semi fluid mixture of cells amino acids, sugars, salts,

organic & inorganic materials• Produced by seminal vesicles prostate gland and

Cowper’s glands.• Ejaculation of human males: 2-6 ml with 100-150 million

sperm cells per ml • Genetic disorder, drug abuse, exposure to certain

chemicals, etc. may decrease sperm count• Appears yellowish-white crust when dry



Sperm cells (spermatozoa)• First co-discovered by van leewenhoek in 1679 (he

identified the first living cell) • Hartsoeker co-discovered sperm cells

– Drew what he hoped to find (preformed human-“homunculus”) (1694)



Hartsocker – drawing “homunculus”



Sperm cells (spermatozoa)• First co-discovered by van leewenhoek in 1679 (he

identified the first living cell) • Hartsoeker co-discovered sperm cells

– Drew what he hoped to find (preformed human-“homunculus”) (1694)

• About 55 m in length (90% is tail)• 3 main parts of the sperm cell

– Head• Contains the nucleus (DNA)• Acrosome: anterior portion that contains enzymes to

penetrate the cell wall of the ovum during fertilization– Midpiece (attaches head & tail) contains the mitochondrion

(provides energy for flagellated movement)– Tail—flagellum (means of locomotion)



Sperm Cell Parts



Presumptive Testing for semen: Brentamine Fast Blue Test

• The primary test for SAP (seminal acid phosphatase) activity

• Produces a color reaction—purple color appears within 30 sec to 2 min.

• Sensitive enough to produce a positive result with a dilution of 500 times

• 2 step procedure– Sterile water on swab/filter paper– Apply swab/filter paper to questioned stain– Reagent added to swab/filter paper– Color change noted

• Several variations exist dependent on the sample



Brentamine Fast Blue Test



Confirmatory Testing for semen• Microscopic Analysis

– Christmas Tree Stain• Most common staining technique

• Specifically developed for sperm cell identification: pink/red heads, blue midpiece, and green tail









• Protein Analysis – p30 (PSA—prostate specific antigen)

• the protein is found only in semen

• uses sample and p30 antibodies—precipitin test



Confirmatory Testing for semen• Protein Analysis PSA

Confirmatory Testing for semen• Protein Analysis PSA



DNA sampling of sperm• Remember that a sperm cell only has 50% of the persons

genes, you need about 80 spermatozoa to generate his complete DNA profile

• PGM, Glycoxalase, Peptidase A are the identifiable enzymes in semen. (Allows one to narrow down rapist’s identity, but not as specific as blood.)



Collection of Rape Evidence• Has intercourse actually occurred? Semen inside of

vagina, physical injuries (on victim)• Observe crime scene for signs of struggle• What needs to be collected?

– Clothing - all (even shoes) – Bedding– Swabs - vaginal, anal, nasal and oral– Combings (head and pubic area) - take exemplars also– Blood - samples and exemplars– Saliva- samples and exemplars– Fingernail scrapings

• If a suspect is taken into custody:– Take suspect’s clothing and examine for trace evidence

(fiber/hair)– Examine suspect’s body for signs of a violent struggle

(fingernail scratches, bite marks, etc.)– Hair and blood exemplars should be taken (legally)



Saliva • No specific test for saliva, but tests rely primarily on the

presence of alpha-amylase (found in animals). [beta-amylase is found in plants]– 2 DNA loci in humans found on chromosome 1:

• AMY1 (codes for amylase found in saliva, breast milk, perspiration)

• AMY2 codes for amylase found in pancreas, semen, and vaginal secretions

• Limitation for testing is the presence of proteins from other body fluids may give false positives.

• If DNA testing is important, it may be better not to consume the sample with body fluid testing (i.e. stamps, envelope flaps, bite marks).



Saliva


Biological Fluids – SalivaBiological Fluids – Saliva



Starch-Iodine Test• Earliest test devised in 1881• Iodine in starch (blue) + amylase color changes

(subsides)• Difficult for locator of stains• No way of quantifying amylase (just relative proportion)



Starch-Iodine Test



Phadebas Reagent • Starch is linked to a dye molecule to form a coloration

(light blue)• Press Test

– Phadebas solution (dissolve tablet in dH2O) and spray on filter paper (allow to dry for storage)

– Wet paper (if necessary) & outline paper on sample for reference

– Press onto the sample with a weight and observe 40 min.• Test Tube

– Variation of test involving liquid samples, centrifugation and analysis of supernatant liquid.

– The coloration level is proportional to the amount of saliva, which can be measured by a spectrophotometer.



Phadebas Reagent – Press Test


Biological Fluids – Blood/SweatBiological Fluids – Blood/Sweat



Urine• Comprised of mostly water and salts• Cellular components are epithelial cells from the urinary

tract (RBCs & WBCs)– Low cell concentrations in urine– Low rate of success in DNA testing (high volumes are better

b/c they can be centrifuged)



Urine (continued)• Identification relies on identifying both urea and creatinine,

which are present in high levels (found in other body fluids also).

• Testing for urea: – Urea + enzyme (urease)ammonia + CO2– Ammonia is detected using an indicator such as Nessler’s

reagent (mercuric iodide or potassium iodide) or DMAC (p-dimethylamino-cinnamaldehyde)

• Testing for creatinine– Sample: + picric acid/toluene (or benzene)creatinine picrate

(creatinine) (colored product)


Biological FluidsBiological FluidsFeces• Solid waste products (undigested food residues, mucosal cells

& bacteria)• Identifying characteristics:

– Stains characterized by a green-brown color (due to bile pigments & products)

– Distinct odor caused by the breakdown of amino acids by bacteria

– Presence of undigested components of foodstuffs– Identified by the detection of urobilinogen (& urobilin

products) - pigments (not present in herbivores) Sample: + Alcoholic zinc acetate urobilin-zinc salt + urobilin

– Urobilin-zinc salt fluoresces bright apple green w/ Long wave UV light.

• DNA testing– Has been unsuccessful due to large amounts of bacteria &

bile products– mtDNA has proven more successful


(urobilinogen)


Vomitus• No tests present for vomitus

– Test for low pH from stomach acids– Amylase– Identification of foodstuffs

• May help to determine time since death if the details of the last meal are known)


Body Fluid Stains – ClothingBody Fluid Stains – Clothing



Vaginal Secretions• Usually identified based on glycogenated epithelial cells

– Not unique to vaginal tract– These cells are absent in prepubescent and post menopausal

females • Periodic acid-Schiff (PAS) reagent stains the glycogen in

the cytoplasm a bright magenta• Testing may consume large amounts of DNA evidence


Biological Fluids – Semen/VaginalBiological Fluids – Semen/Vaginal


erology s s by m. hoffmeister & h. montgomery. identification & character of blood and...

Documents

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identification of blood

blood analysis

analysis of blood

characteristic of blood

characterization of

traces of blood

catalyst catalytic color