Evidence Screening

Question 1

Locard’s Exchange Principle

Answer 1

Edmund Locard developed the first crime lab in France in 1910. He stated that whenever two objects are in contact there will always be transfer of material from one to the other.

Question 2

Modes of transfer

Answer 2

Primary transfer: direct contact (hand shake)

Secondary transfer: indirect contact through a medium (person A touches something then person B handles)

Can extend beyond secondary transfer

Question 3

Why would the amount of DNA on an item be expected to be higher?

Answer 3

With more handling or time spent in contact, on a rough surface, wet stain, higher starting amount

Question 4

Why would the amount of DNA on an item be expected to be lower?

Answer 4

With multiple transfers, dried stains, smooth surfaces, lower starting amount

Question 5

What is the purpose of evidence screening?

Answer 5

To identify bodily fluids and localize sources of DNA. Any cell with nuclear DNA can be used for typical DNA testing.

Question 6

Presumptive vs Confirmatory tests

Answer 6

Presumptive tests: fast and easy, screen large areas, have false positives, performed before more specific test

Confirmatory: take longer, unique to the body fluid

Question 7

What does blood consist of?

Answer 7

Cellular and plasma components

Question 8

What are the three things that make up the cellular (45%) component of blood?

Answer 8

1) Red blood cells: 99% of cells in blood, lack nucleus, have a 3-4 month lifespan, contain hemoglobin, distribute oxygen
2) White blood cells: nucleated cells that contain DNA, extracted to DNA profiles, are part of the immune system which is involved in antibody production and phagocytosis
3) Platelets: non-nucleated cell fragments that play an important role in blood clotting

Note: white blood cells and platelets only account for 1% of the cells in blood

Question 9

Describe the plasma (55%) component of blood.

Answer 9

Liquid portion of blood, yellow fluid, contains water, salt, vitamins, proteins, etc.

Question 10

Blood identification

Answer 10

Involves the red blood cells which contain the protein hemoglobin (consisting of four subunits responsible for transporting oxygen from lungs to cells and CO2 from cells to lungs). Hemoglobin exhibits peroxidase activity and is not found in any other bodily fluid unless that fluid contains blood.

Question 11

Tell me about the appearance of blood.

Answer 11

Red liquid, brown dried, yellow washed, but colour is not used to determine the age of the bloodstain.

Question 12

List the five presumptive blood tests.

Answer 12

Phenolphthalein (Kastle-Meyer), Leucomalchite green, Hemastix, Luminol. and oxygen-reduction reactions (redox)

Question 13

What are redox reactions?

Answer 13

Oxygen-reduction reactions where the oxidation of a molecule results in the loss of electrons, and the reduction of a molecule results in the gain of electrons

Question 14

What is the difficulty with presumptive blood identification tests?

Answer 14

Presumptive tests rely on the peroxidase-like activity of hemoglobin, so the test must be resistant to false positives with cleaning supplies, fresh plant compounds, and metals.

Question 15

Describe the Kastle-Meyer test; its process, chemicals used, advantages, and disadvantages

Answer 15

• Need to perform a chemical test since some substances may appear to be blood, but are not (ketchup, rust, etc)
• Rub test – a piece of filter paper is rubbed against a suspected bloodstain, transferring some of the material to the paper
• Reagents are then applied to the paper in the specific order, not adding the chemicals directly to the bloodstained exhibit!
  1) Methanol – makes the test more sensitive by “cleaning up”, improving access to hemoglobin
  2) Phenolphthalin – if a pink colour develops at this step, it has been oxidized by something in the sample that is not blood (that is why you should wait briefly before adding H2O2)
  3) H2O2 – is catalyzed by haemoglobin (peroxidase activity), releasing the oxygen that oxidizes phenolphthalin
• Monitor paper for 10 seconds
  If blood is present a pink colour will develop
• After 10 seconds test is stopped because oxygen in the air will turn the paper pink

Advantages: very sensitive and very fast, only use a small portion of stain so can have sample left for DNA analysis

Disadvantage: not specific to human blood

Question 16

Describe the luminol test

Answer 16

This is the test often seen on tv. Luminol reacts with H2O2 and hemoglobin to emit light, which means it can only be seen in the dark which is a bit of a down side, along with the fact that the entire stain is often exposed to the chemical.

Question 17

What are some redox assay issues?

Answer 17

Presumptive tests are not specific to blood. If strong oxidants are present it can cause a positive reaction without the presence of heme (copper, nickel, bleach). Plus, plant peroxidases (many plants contain peroxidases that can catalyze the reactions and lead to false positives)

Question 18

What is the hemochromogen test?

Answer 18

It is a confirmatory blood test (aka Takayama crystal assay) based on the crystallization of hemoglobin that confirms the presence of blood, but it cannot distinguish between human and animal blood.

Question 19

What is the hemochromogen reaction?

Answer 19

A bloodstain is treated with pyridine and glucose under alkaline conditions which will form crystals of pyridine ferroprotoporphyrin. The appearance of pink/red feathery crystals indicates the presence of blood.

Question 20

What are the advantages and disadvantages of hemochromogen?

Answer 20

Advantages: test is specific and no known false positives

Disadvantages: reagent is carcinogenic, requires a sufficient size stain and the sample tested is consumed, requires microscope, reagent, fume hood, not specific for human blood

Question 21

What happens if a DNA profile is not obtained from a bloodstain?

Answer 21

The blood is not human, or is degraded or inhibited. A follow up test can be performed to confirm human origin.

Question 22

What is the ABAcard hematrace?

Answer 22

It’s a confirmatory immunological test for human blood.

1) If human hemoglobin is present, its antigens bind to the mobile monoclonal antihuman Hb antibodies (containing a dye), forming a mobile antibody-antigen complex which migrates to the “T” area.
2) The “T” area contains stationary polyclonal antihuman antibodies which capture the mobile antibody-antigen complex, forming an antibody antigen-antibody sandwich
The antibodies are labeled with a pink dye, forming a pink line in the “T” zone, indicating that human hemoglobin is present.
3) The internal positive control in the “C” area; stationary anti-immunoglobulin antibodies bind the excess antihuman Hb monoclonal antibodies that do not bind to the antibodies in the “T” zone, forming a pink line.

Question 23

How to interpret the ABAcard?

Answer 23

Positive Result: one line at both the ‘T’ and ‘C’ areas

Negative Result: one line at only the ‘C’ area

Invalid Result: no line at the ‘C’ area

Question 24

Define oligospermic.

Answer 24

Low sperm count.

Question 25

Define aspermic.

Answer 25

No sperm count, often as a result of a medical condition, disease, chemo, or vasectomy

Question 26

What is the average ejaculate volume?

Answer 26

Roughly 3.5mL, containing 60-100 million spermies

Question 27

What is seminal plasma?

Answer 27

It constitutes 90% of semen, and is an extracellular fluid that provides a medium and vehicle for the sperm. It originates in the male accessory organs of reproduction (e.g. prostrate) and is a mixture of fluid, including enzymes and proteins.

Question 28

What is the cellular fraction in sperm?

Answer 28

It’s sperm and it constitutes 10% of semen.

Note: in a vasectomy the dude still produces an ejaculate with seminal plasma but no cellular fraction.

Question 29

Whats in the sperm head?

Answer 29

It largely consists of the nucleus which contains the DNA. This is the part that forensics looks at for a DNA profile. The head also has a dark acrosome on the tip.

Question 30

Tell me about the neck and tail of the sperm.

Answer 30

The neck is the most fragile component; a narrow region that connects the head to the tail. The tail provides motility to sperm through the whipping action of the axial filament. A tail may or may not be present and may detach over time or if exposed to different environmental insults (tail presence indicates a recent event).

Question 31

Presumptive tests that test for semen.

Answer 31

Acid Phosphatase (AP) also known as Fast Blue (FB) Test, p30 Test (PSA Test)… if these tests are positive then it is POSSIBLE that semen may be present.

Question 32

Confirmatory test for semen.

Answer 32

Microscopic examination… if HUMAN spermatozoa is observed, semen IS present

Question 33

Describe the acid phosphate test for semen; the reaction, results, advantages, and disadvantages.

Answer 33

Acid phosphate is produced in the prostate and found in very high concentrations in semen. A chemical screening test is done to localize area(s) of interest, but does not confirm the presence of semen. It’s based on the activity of the enzyme, acid phosphatase.

Reaction: in the presence of the substrate sodium αnaphthyl acid phosphate (salt), the enzyme acid phosphatase will cause the release of naphthol, which reacts with the dye to form a purple colour.
One step procedure: add Fast Blue Reagent (contains both the salt and dye), monitor for colour formation

Results:
Positive reaction: purple colour within 1-2 mins, faster time means more acid phosphatase present
Negative: not colour change, or any colour that is not purple

Advantages: reasonably sensitive, fast and easy, non-destructive, can screen large exhibits (localize areas for follow up), can detect seminal fluid from vasectomized and azoospermic males (no spermatozoa but acid phosphate is still present in ejaculate)
Weaknesses: not specific, false positives exist, false negative – not as sensitive as other tests, can’t be used on laundered items

Question 34

Protein p30 (aka Prostate Specific Antigen PSA) detection test? False negatives, false positives, and advantages/disadvantages?

Answer 34

Secreted by prostate and found in all semen, this test does not confirm, and works just like the ABAcard. Pink dye is present in the card, if the human antigen is present it binds to the dye and makes a pink line in the test area. Immobilized antibody is present in the control area to bind with the pink dye already in the card to make the test valid. However, excessive antigen or environmental insults can cause a false negative, while urine, breast milk, blood from prostate cancer patients, etc. can cause false positives.

Advantages: very sensitive, fast and easy, commercially available, can detect seminal fluid from vasectomized and
azoospermic males (P30 still present in ejaculate)

Disadvantages: false positives, cant be used on laundered items

Question 35

What is microscopic examination?

Answer 35

Used to spot human-specific spermies, a “christmas tree” stain can be used to stain cytoplasm green and sperm head red.

Question 36

Slide treatment for finding spermies from a vaginal swab or cut of clothing

Answer 36

Pretreatment with sodium dodecyl sulphate (SDS denatures proteins) and proteinase K (ProK digests proteins)