Midterm

Quality Assurance

implementing strategies or procedures to ensure the reliability, accuracy, and consistent of lab results

System put in place to prevent errors or contamination

Proactive, preventative system focused on establishing processes and procedures like SOPs and training

Quality Assurance means

building quality in

preventative

Quality control

reactive

reactive processes that use routine checks to identify, measure, and correct deficiencies and confirm that the quality system is working effectively

day to day procedures to make sure QA is being maintained

Quality control means

checking quality out

reactive

What factors determine the correctness and reliability of tests? - QA

• Human Factors

• Organization Structure

• Physical facilities and environmental conditions

• test methods and method validation

• equipment

• handling of test items and standards

• measurement standards

• technical records and reporting

• SOPS

Human Factors - QA Factor

Staff needs to be suitably qualified and experienced for the activities they are required to perform. Includes

• appropriate training and competency tests

• regular monitoring of staff performance (periodic proficiency testing or evaluation of QC data produced by them)

• records kept that detail and explain everything

Organization Structure - QA Factor

People need to be trained to have a background for their job and there must be a hierarchy of job roles

• should have supervisors with analysts under them

• everyone is laid out having different jobs and letting everyone know who is who, so they know who to report to

Physical Facilities and Environmental Conditions - QA factor

lab and office accommodations must be suitable in construction and size to perform the range of activities undertaken

• Environmental conditions must be appropriate (temperature, humidity, lighting, dust free, clean and sterile lab, etc.)

• space should allow for separation of noncompatible activities

  • ex. DNA extraction separated from DNA amplification, or firearms testing separated from GSR analysis area

    • DNA labs mostly have one direction of movement to ensure excess contamination does not occur

  • space must be secured with controlled access

Test Methods and Method Validation - QA factor

It must be possible to demonstrate, through validation or verification records, that all analytical methods used are fit-for-purpose

• precision and accuracy of test methods

• LOD and LOQ

• specificity, linearity, range

• repeatability and reproducibility

• life cucye of method validation

Precision vs Accuracy of test results

precision - all runs give same results

accurate - results are correct

repeatability vs reproducibility

repeatability - if I do the procedure 10 times it will be the same all 10 times

reproducibility - if multiple people do the procedure, the results are the same

Equipment - QA factor

all equipment and instrumentation needs to meet specifications consistent with intended purpose, be suitably maintained to ensure satisfactory performance, and be calibrated against recognized standards where relevant

• maintenance

• safety

• calibration checks

• records maintained and monitored

handling of test items and standards - QA factor

COC and retention of retesting - must be operation procedures in place to determine how long we are keeping evidence

lab must have and apply procedures to protect samples from loss, contamination and degradation

Measurement Standards - QA factor

• chemical or physical standards used for calibration or validation purposes

• documented use and storage of these standards to preserve integrity and should be certified by an external body

Technical Records and Reporting - QA factor

Should include everything that has to do with evidence

may include

• handwritten notes

• worksheets

• instrument print outs

• photographs or digital images and administrative paperwork

SOPs - QA Factor

Must be detailed, step-by-step instructions for routine tasks to ensure consistency and accuracy

Should be

• detailed and specific

• consistency

• comprehensive

• accessible

• compliance

Internal QC Procedures

• Analysis of reagents or subtract blanks - make sure reagent itself isn’t giving wrong detection

• analysis of previously analyzed samples

• replicate analysis of samples

• analysis of interlaboratory “blind” samples

5% of samples analyzed should be a QC sample - control or blanks

External QC

interlaboratory collaborative trials or samples exchanged to see if everyone gets the same results

Proficiency tests

“blind” test submitted to an analyst for processing and the analyst does not know the expected outcome

• serves as QC for individual and the lab for validity of test methods, equiptments, reagents, etc

Horizontal Internal Audits

cover a single aspect of a lab’s quality system to check on compliance against documented procedures

vertical internal audits

follow the progress of a case from receipt through examination, sample analysis, interpretation of results, reporting and case finalization

external audits

audit undertaken by an accreditation agency or by representatives from another lab

ISO 17025

international std for competence of testing and calibration labs setting general requirements for quality management system

ISO 17020

international std designed to ensure that "inspection bodies" carry out competent, independent, and impartial inspections to provide their clients with info in terms of conformity with regulations, stds, or other specifications

What could happen without a robust QA/QC system?

• evidence may be contaminated

• reports may be inaccurate

• findings may lack credibility

Direct Evidence

evidence that if accepted would prove a fact without any need of an inference to be drawn from it

Circumstantial or Presumptive Evidence

suggests a fact by implication or inference, requires reasoning to connect it to a conclusion

Evidence must be…

relevant, probative, and admissible

Physical Evidence

•  any tangible object that can connect a suspect to a CS

  • Ex) Dirt, gun, bullets, fingerprint, fibers, sand, soil, tool marks, ammunition, firearms, glass, footprint, documents

Biological Evidence

•  comes from living organisms and contains DNA or other organic material

  • Ex) blood, semen, saliva, urine, sweat, skin cells, hair, organs, bone

Biological Evidence Processing Steps

1. Identification - determining type of body fluid and if from human or animal

2. individualization - linking the biological evidence to a specific individual

3. reconstruction - using biological evidence to understand the sequence of events at a CS

Control Samples

samples, swabs or cuttings from material alone the biological evidence is on

used to confirm the results of the test performed

reference samples

Samples collected from all individuals who may be linked to the CS to act as eliminatory or comparative evidence

What must biological evidence packing be labeled with?

• evidence description

• evidence source - the origin, where it was, when collected

• COC

• unique identifier number

• can indicate what processing is needed, health hazards, required or preferred storage conditions

When should biological evidence be wet or dry?

Blood-draw samples or some contents of a SA kit are the only evidence that remain in liquid form, everything else should be dried properly to be stored and then tested in the future.

• limits microbial growth that will impede DNA

Packaging Types

Evidence Bags

• More bulky evidence such as clothing and bedding

Boxes

• sharps box - knives, or gun box

Evidence envelopes

• small items of evidence - swabs, sm fibers/trace evidence, cigarette butts

Short-Term Storage Conditions for biological evidence

Holds evidence up to 72-hours

• liquid blood - refrigerated

• Urine - Frozen

• Dry Biological Stained Item - Temperature controlled

• Wet Bloody Item (cannot be dried) - frozen

• Bones - can be frozen, temp controlled, or room temp

• Hair - temp controlled

• swabs with biological material - wet: refrigerated, dry: temp controlled

• Vaginal Smears - Temp controlled

• feces - frozen

• buccal swabs - temp controlled

Long-Term Storage Conditions for biological evidence

Hold evidence more than 72 hours

• liquid blood - refrigerated

• Urine - Frozen

• Dry Biological Stained Item - Temperature controlled

• Bones - temperature controlled

• Hair - temp controlled

• swabs with biological material - dry: temp controlled

• Vaginal Smears - Temp controlled

• feces - frozen

• buccal swabs - temp controlled

• DNA extracts - liquid: Frozen

COC Documentation Should Include

• Description of the evidence

• Unique case identifier (case #)

• Where the evidence was collected

• Where the evidence was stored

• Who was in possession of the evidence and for what purpose

• What was done to the evidence (analysis or re-packaging)

Numerical identifiers for cases and evidence items

• Case # and evidence # always associated

• Case # always the same, evidence # tends to change

Biological Evidence Disposal

1. Incineration - burning, destroys and contaminates evidence

2. Thermal Treatment - heat to destroy pathogens

3. Chemical Treatment - chlorine in form of sodium hypochlorite solution or bleach or chlorine dioxide gas

   1. contaminating the evidence

SAFE - Sexual Assault Medical Forensic Examination

• An examination conducted by a clinician on a patient who has experienced SA done by a SANE

Need to be coordinated approach, victim-centered care, informed consent, confidentiality, reporting to law enforcement (up to victim), payment waved by VAWA

SANE - Sexual assault nurse examiner

• Qualification for forensic nurses who have received special training to conduct SA evidentiary exams for rape victims in the US

SAK - Sexual assault kit

• Contains

  • Kit container

  • Instruction sheet

  • Forms

  • Materials to collect and preserve evidence

    • Patients clothing/material dislodged from clothing

    • Foreign material on patients body

    • Fingernail scrapings/cuttings

    • Hairs

    • Vaginal/cervical swabs

    • Penile swabs

    • Anal/perianal swabs

    • Oral swabs

    • Body swabs

      • all skin swabs collected with light touch, in circular motion for strangulation, oral contact (kissing, licking, biting, sucking), ejaculation on skin, touch DNA by holding skin

    • Known blood/saliva sample, or buccal swab for DNA analysis and comparison

• Must be very careful while processing SAKs - need to wear proper PPE, don’t sneeze or breathe too closely to kit

Serology

The study of serum - the watery liquid part of blood that holds the water, proteins, hormones, lipids, antibodies and antigens NOT RBC, WBC or platelets

Forensic Serology

Detection and Identification of serum or serological fluids in a legal context

identification of body fluids in a forensic context (not only analyzing blood, other bodily fluids)

Ways to Identify a potential Biological Stain

• 1st step - oblique and ALS

• 2nd step - Presumptive Testing

  • color tests

• 3rd Step - Confirmatory Testing

  • immunologic testing

Alternate Light Source

Can make a stain or substrate fluoresce if the correct wavelength is used.

Mainly used for locating and enhancing suspected stains.

Semen, Saliva, urine will glow under certain light because they will absorb the original light and reemit the light at a lower energy, lower wavelength EM radiation.

Blood absorbs light making it look darker

Presumptive Testing

Make use of a target chemical to establish the possibility that a specific body tissue or fluid is present to narrow down possibilities and help decide what tests to do next.

Presumptive tests INDICATE the presence of a bodily fluid

Should be quick, simple and safe

most rely on color changes

Confirmatory Testing

Used to IDENTIFY the specific biological material, which can then be typed.

Always after a presumptive test, to conclusively identify a substance because it costs more, requires additional equipment, takes longer, has less false positives.

Most rely on interactions of antibodies and antigens

Confirmatory tests IDENTIFY the presence of a bodily fluid

Color Tests

Most presumptive tests

Either the chemical structure changes through an electron transfer losing a hydrogen atom, changing the structure and allowing the substance to absorb and reflect light differently

OR the chemical reaction releases a colored molecule from binding interactions with the substrate’s molecular structure and relies on fluid-specific enzymes.

Immunologic Testing

Often used for confirmatory testing

Lab techniques that detect antigens or antibodies using specific antigen-antibody binding.

• Antigen: Protein/polysaccharide structure recognized by the immune system.

• Antibody: Immune protein that binds specifically to an antigen.

• Confirmatory test: Detects an antigen unique to one body fluid.

Could you have a negative for a presumptive and a positive for a confirmatory?

Yes, because there are false negatives and confirmatory tests are more sensitive.

3 major components in blood

1. RBC - no nucleus

   1. possess the pigment molecule of hemoglobin to give red coloration

   2. transport O2 and CO2

2. WBC - have a nucleus

   1. responsible for immune defense - secrete antibodies that react to foreign components in the body

3. Platelets - no nucleus

   1. responsible for clotting mechanism

Plasma

Majority of the composition of blood - 55%

45% = RBC and 5% = WBC and platelets

ABO Blood system

• RBC and WBC have antigens on surface recognized by antibodies

Key rule: You produce antibodies against the A/B antigen you do NOT have.

• Type A: A antigens + anti-B antibodies

• Type B: B antigens + anti-A antibodies

• Type AB: A + B antigens + no antibodies

• Type O: No A/B antigens + anti-A + anti-B antibodies

Blood typing test: Add a known antibody to the blood. If the matching antigen is present, agglutination (clumping) occurs.

Presumptive Blood Detection Reaction

Presumptive blood tests detect hemoglobin by using the peroxidase-like activity of heme iron to oxidize a colorless dye, producing a color or light.

Reaction:
Blood (heme/Fe) + H₂O₂ + colorless dye → oxidized colored dye

How it works:

1. Heme contains iron (Fe).

2. Iron reacts with H₂O₂, becoming activated.

3. Activated heme removes electrons from the dye → oxidation.

4. Oxidized dye changes structure → visible color/light.

Remember:
Blood → hemoglobin → heme → iron → reacts with H₂O₂ → oxidizes dye → color/light

Kastle-Meyer Test

Presumptive Color Test for Blood

all steps done directly on the swab

Heme catalyzes H₂O₂ oxidation of phenolphthalin → pink phenolphthalein = positive result

Kastle-Meyer Steps

1. Apply phenolphthalin to the suspected stain/swab.

2. Add H₂O₂.

3. Heme acts as a peroxidase-like catalyst.

4. Heme helps break down H₂O₂ → reactive oxygen species.

5. Reactive species oxidize phenolphthalin → phenolphthalein.

6. Pink = positive.

Key: Heme + H₂O₂ → oxidation → pink.

Leucomalachite Green Test

Presumptive Color Test for blood

Heme catalyzes oxidation of leucomalachite green → green malachite green = positive result

Leucomalachite Green Steps

1. Apply leucomalachite green to the suspected stain.

2. Add H₂O₂.

3. Heme iron acts as a peroxidase-like catalyst.

4. Heme catalyzes oxidation of leucomalachite green.

5. Leucomalachite green → malachite green.

6. Green = positive.

Key: Heme + H₂O₂ → oxidation → green.

Luminol Test

Presumptive Color test for Blood

Heme catalyzes H₂O₂ reactions that oxidize luminol, producing a blue glow if positive

Luminol Steps

1. Mix luminol + NaOH + H₂O₂.

2. Heme iron catalyzes the breakdown of H₂O₂.

3. OH⁻ reacts with luminol → luminol dianion.

4. Dianion reacts with oxygen.

5. Oxidation produces blue light.

6. Blue glow = positive.

Key: Heme + luminol + H₂O₂ → blue chemiluminescence.

Hematrace Test

Confirmatory Test for Blood

Antibodies on a test strip bind human hemoglobin antigens, producing a positive result.

Hematrace Steps

1. Apply the suspected blood sample to the test strip.

2. Sample encounters antibodies specific to human hemoglobin.

3. Human hemoglobin binds to the antibodies.

4. Antigen-antibody reaction produces colored lines.

5. Positive = human hemoglobin detected.

Key: Antibody + human hemoglobin → positive test.

Takayama Test

Confirmatory Test for Blood

Blood forms pink, feathery hemochromogen crystals visible under a microscope.

Takayama Steps

1. Add pyridine, glucose, and NaOH to the suspected blood.

2. Hemoglobin is reduced to hemochromogen.

3. Hemochromogen forms crystals.

4. Examine crystals under a microscope.

5. Pink, feathery crystals = positive.

Key: Takayama → pink, feathery hemochromogen crystals.

Teichmann Test

Confirmatory Test for Blood

Blood forms brownish-black, rhomboid hemin crystals under a microscope.

Teichmann Steps

1. Add acetic acid + NaCl to the suspected blood.

2. Gently heat the sample.

3. Iron in hemoglobin reacts with chloride → hemin.

4. Allow the solution to cool.

5. Examine under a microscope.

6. Brownish-black, rhomboid crystals = positive.

Key: Teichmann → brown/black rhomboid hemin crystals

What is the major limitation of Kastle-Meyer and Leucomalachite Green?

They detect hemoglobin, but do not confirm that it is human blood.

Major advantage for luminol

It is highly sensitive, non-destructive, and can detect old or washed blood.

Saliva

• Produce 1-1.5L/day

• most common biological evidence found next to blood

• identification of saliva largely based on detecting the presence of amylases

  • salivary alpha-amylases

  • beta-amylase

  • gamma-amylase

Starch-Iodine Test

Presumptive color test for saliva

Salivary amylase breaks down starch, preventing the starch-iodine complex from forming.

yellow/brown color = saliva present, blue = saliva absent

Starch-Iodine Steps

1. Add starch to the suspected sample.

2. Add iodine.

3. If starch is present, starch + iodine → blue.

4. If saliva is present, α-amylase breaks down starch.

5. Starch is no longer available to react with iodine.

6. Yellow/brown = positive for saliva.

Key: Amylase breaks down starch → no blue color.

Phadebas Test

Presumptive Color test for saliva

α-amylase digests starch microspheres, releasing a water-soluble blue dye. it works by salivary α-amylase breaks down starch microspheres, releasing the blue dye.

Phadebas Steps

1. Add the sample to Phadebas reagent.

2. Reagent contains starch microspheres with blue dye.

3. Salivary α-amylase digests the starch.

4. Digestion releases the water-soluble blue dye.

5. Dye diffuses into the solution.

6. Blue = positive for saliva.

Key: Amylase digests starch microspheres → blue dye released

SALIgAE

Presumptive Color Test for Saliva

A yellow color indicates the presence of saliva.

Urine/feces can cause cross-reactivity, and blood can interfere with interpretation.

SALIgAE Steps

1. Add the suspected sample to the SALIgAE reagent.

2. Reagent reacts with components associated with saliva.

3. Allow the reaction to develop.

4. Observe the resulting color change.

5. Yellow = positive for saliva.

Key: Yellow color = positive.

RSID-Saliva

Confirmatory Antibody Test for Saliva

Uses antibodies specific to saliva to detect saliva antigens.

RSID-Saliva Steps

1. Apply the suspected sample to the RSID-Saliva test strip.

2. Sample encounters saliva-specific antibodies.

3. Saliva antigen binds to the antibodies.

4. Antigen-antibody complex produces a colored test line.

5. Positive line = saliva detected.

Key: Saliva antigen + specific antibody → positive test.

What enzyme is primarily used to detect saliva?

α-Amylase, which breaks down starch.

Semen

Thick, viscous liquid

Male ejaculation is a mixture of 5-10% sperm and 90-95% seminal plasma

Healthy human male can release 2-6 mL per ejaculation

Acid Phosphatase Test

Presumptive Color Test for Semen

1. Apply AP substrate to the stain.

2. AP breaks down the substrate.

3. Product reacts with Fast Blue B color developer.

4. Blue/purple = positive for AP.

Key: Semen contains high concentrations of acid phosphatase.Test for Semen

Sperm Search

Confirmatory microscopic test for semen

1. Prepare the suspected semen stain.

2. Examine the sample under a microscope.

3. Look for spermatozoa.

4. Sperm present = confirmation of semen.

Key: Provides direct visual identification of sperm morphology.

P-30 PSA

Confirmatory Antibody Test for Semen

1. Apply the suspected stain to the ABA card/test.

2. Add antibodies specific to PSA/P-30.

3. PSA binds to the antibodies.

4. Antigen-antibody reaction produces a positive result.

Key: PSA/P-30 = prostate-specific antigen → semen.

Role of Forensic Serology

to aid in the corroboration of the scenario or refute the scenario based on what we find

Reports follow KISS

Keep It Simple, Stupid

The criminalistics value of hair

Can get information on how, when, who

• how the hair was cut

• when in the life cycle was the hair lost

• who - if have root, can do DNA analysis

Hair Follicle

•  is the living, tunnel-shaped structure in the skin that grows hair, includes the root

  • A downgrowth or invagination of the epidermis into the dermis

  • Part located inside the skin and where the hair grows

  • Hair follicle has

    • Hair bulb which holds the

      • Root

Hair Shaft

•  visible, nonliving strand that emerges above the skin

  • Part of hair we see

Hair Cycle Phases

Anagen

Catagen

Telogen

• stages in alphabetical order

Anagen Stage

• 1st Stage

• Cell division

• Formation of keratin - main proteins in hair that gives hair structure

• Phase lasts 2-7 years

• Determines hair length

• Growth phase

Catagen Stage

• 2nd stage

• Cell division slows - bulb part isnt as big as in anagen stage

• Follicle shrinks and starts to detach from bulb

• Lasts about 2-3 weeks

Telogen Stage

• Final stage

• Hair growth stops and old hair eventually falls out

• Lasts around 3 months before cycle restarts

What stages can you get DNA from the hair?

Anagen and Catagen bc still have the root and living cells in the hair

90% of human head hairs in anagen or catagen growth phase

What gives hair color?

melanocytes in the hair follicle are responsible for synthesizing melanin

• with age, production of melanin stops - gray hair appears

• vitiligo or albinism involve melanocyte dysfunction or absence

What stage hair do you usually find at crime scenes?

Telogen

3 layers of the hair shaft

cuticle - outer layer of overlapping cells

cortex - provides strength and color, majority of the hair

medulla - core, sometimes absent

• can be absent, fragmented, continuous

Cuticle → cortex → medulla

Level 1 Hair examination

recognition and separation of human and animals hair

based on shaft-profile

Looking at

• Hair type

• hair length

• root shape

• color

• medulla

• cuticle

Hair Type - Level 1 exam

• Animals have 2 types of hair - outer hair or under coat hair present in different shapes, don’t see in human hair

• Format of hair can see differences in shape