SEROLOGY SEROLOGY The science of body fluid identification

SEROLOGY

SEROLOGY The science of body fluid identification and characterization (typically blood, semen, saliva, and urine)

Serology • Examination of blood, semen, saliva, and urine through the use of presumptive tests and confirmatory tests

Serology starts with the suspect’s or victim’s clothing, weapons, and other evidentiary items

Cuttings or swabbings are taken from evidence

Blood is composed of • cells – red blood cells (erythrocytes), white blood cells (leukocytes), and platelets; cells make up 45% of blood content • plasma – fluid portion of un-clotted blood; plasma makes up 55% of blood content • Where is DNA? white blood cells

Blood • Red blood cells – about 5 million per microliter • White blood cells – 2, 000 to 7, 000 per microliter; present in much smaller concentration than red blood cells, but this is where we get DNA from • Red blood cells have no nucleus and, therefore, no nuclear DNA

Blood - “possibly” Presumptive Test: a chemical test that uses color change to detect the possible presence of blood – Fast Reaction – Not Specific

Phenolphthalein Test • Hemoglobin (red blood cell protein that transports oxygen) possesses peroxidase -like activity that oxidizes phenolphthalein yielding a bright pink color. • hemoglobin + phenolphthalein reagent + hydrogen peroxide = bright pink color • Presumptive test for blood, but cannot rule out other sources – false positives (e. g. copper, broccoli, fruit juice) • Not human specific (e. g. reacts with animal blood)

Hemastix Test • Reacts with blood hemoglobin – Peroxidase-like activity • Positive reaction in seconds • Not human specific • Hemoglobin

Hemastix Test

Presumptive Tests for Blood Presumptive Screening Tests For Blood Test Indicator Sensitivity Benzidine Blue color Tetra-methylbenzidine Blue color o-Tolidine Dark green + + + + + Phenolphthalein Pink color + + Safe Leucomalachite green Green color + + Relatively safe Luminol Fluorescence + + + Probable carcinogen * tests are not human specific Comment Carcinogen Probable carcinogen

Luminol is used by forensic scientists to detect trace amounts of blood left at crime scenes.

Blood Enhancement with Luminol/Fluorescein Drain grate in holding cell – blood visualized with luminol

Blood Enhancement with Luminol/Fluorescein Bloody shoeprint and other bloodstains on flooring

Blood Identification • Confirmatory test for blood uses the formation of crystals to indicate the presence of blood. Takayama Crystals or Hemochromogen Crystals

Origin Test The Ouchterlony test uses antibody reactions to determine if blood is human.

Semen composed of • spermatozoa – the male reproductive cell; 100 million cells per ml; 2. 5 – 6 ml per ejaculate (meaning each ejaculate contains up to 600 million sperm cells) • seminal fluid – fluid portion of semen that contains acid phosphatase and PSA (prostate specific antigen) • acid phosphatase – 400 X more concentrated in semen than in other body fluids • PSA or p 30 – unique to semen • Where is DNA? spermatozoa (sperm cells)

Semen - “possibly” • Presumptive Test: a chemical test that uses color change to detect the possible presence of semen Acid Phosphatase Blue color change indicates presence of acid phosphatase – “possibly semen”

Semen Identification • Confirmatory/Origin Determination Test: an antibody reaction test that indicates the presence of human semen through the formation of a precipitant band on the test device Negative Test p 30 test C T S S Positive Test

Semen Identification • Spermatozoa – Christmas tree staining; stains heads of sperm cells red and tails green allowing for microscopic visualization • Prostate Specific Antigen (p 30) – unique to seminal fluid; PSA card Christmas Tree Staining .

Saliva - “possibly” Starch Agar is an all-purpose medium which is flooded with Gram's iodine. The iodine reacts with starch to form a dark bluecolored complex. Any clear area around the sample indicates the breakdown of starch due to the presence of amylase, an enzyme found in saliva. Where is DNA? epithelial cells (cheek skin cells)

Purple top - EDTA After serology testing, samples are prepared for DNA typing

Masks and Caps • Items commonly used by bank robbers to disguise themselves • DNA can be obtained from the saliva left on the mask or from the sweatband of a baseball cap

Chewing Gum • A ring of thieves would steal cars to use as their “get-away” vehicles • DNA extracted from gum (epithelial cells) taken from the ignition switch of the stolen cars matched one of the suspects • He confessed and led police to the others involved

Bandages • Bank robber covered his face with bandages to disguise himself • He threw them away outside the bank • DNA recovered from the adhesive portion matched the suspect

Serology Protocols • • • Date issued and revision number Scope, background Equipment Controls (+ and -) Precautions – work surface, PPE, one item QC procedures – (i. e. test ALS with +) Analytical procedure Limitations Safety References Signatures

Serology Protocols – Positive/Negative Controls • Positive control - a known sample that indicates equipment/reagents are working properly • Negative control – tests for contamination of reagents and materials