Bodily fluids

What are bodily fluids

Liquids ex/secreted by or present within the body at any given time

Excreted- dispelled as waste by te body e.g faeces, vomit

Secreted- transferred from one area of body to another e.g blood/plasma and semen

Forensic applications

Bodily fluids within the human body

Secreted fluids- transferred to victim/scne

Helps forensics determine

how/when the victim died

who the offender was

What can forensic pathology do

Understand the cause, manner and time since death e.g stomach contents/vomit analysis

Forensic value

• Present at various crime scenes

• persistance over time- good for cold cases

• Detection + identification- do DNA analysis- individualization

Why are bodily fluids not always straightforward

Often small quantities

may be a mix of fluids

often cant see them - have to assume their prescence

Detection of fluids

Screening techniques at scene

• Alternative light sources

• Chemical agents (luminol)- doesnt destroy the DNA

Locate fluids ‘in situ’ before collection

Forensic approach following detection- 3 steps

• presumptive tests- indicate potential source

• confirmatory tests- conclusively identify type of biological material

• downstream analyses- individualise DNA

the tests are..

• vary by type of fluid

• non-destructive

• scene based/lab based

• avoid contamination

Collection of fluids

• scene, evidence, and or persons- documentation and sensetive techniques such as swabs, taoes and cuttings

• comparison samples (controls)

• measures to avoid cross contamination

Blood

• the most common bodily fluid encountered- murder assault

• volume recovered varies- drop, stain, pattern

• detectable after cleaning attempts

• various presumptive tests available

Uses of blood for forensic

Who was involved

what happened

when did it happen

what evidence needed from suspect

The nature of blood

• Its viscous- 3 to4 times thicker than water

• Due to its different components:

  55% plasma- water, antibodies, proteins, enzymes

  Inorganic substances-drugs/ alcohol

• 45% cellular material

What are the different types of cellular material in blood

Erythrocytes- rbc’s and are the most common (44%)

Leucocytes- wbc’s and <1% blood volume

Thrombocytes- platelets and cell fragments

Erythrocytes

• Contain haemoglobin

  -Iron containing protein carries O2 and CO2

  -Gives blood its colour

• Shape = flexibility for travel

• Antigens on surface of the- blood type characteristics

Leucocytes

• Contain nuclei= DNA

• Protect against infectious disease + invaders

What are the 5 types of white blood cells

Granulocytes- fight of bacterial infection

Lymphocytes- help maintain healthy immune system

Monocytes

Thrombocytes

• Irregularly shaped, colourless-produced in bone marrow

• Sticky surface- form clots- stops bleeding

• Fluidity of the blood

• Active when circulatory system walls are damaged

Blood types

• Classification of blood based on presence/absence of:

  -antibodies (plama)

  -inherited antigenic substances (RBC’s)

• Blood type inherited from both parents

• Two important systems

  -ABO

  -RhD antigen

What is the ABO system

• Denotes presence of one, both or neither A or B antigens on RBC’s

• Initially discovered by Landsteiner (1901)

• Four main groups - A,B,O and AB

• Phenotypes vary in different populations

What is the Rhesus (Rh) system

• 2nd most important blood group system

• 50 defined blood groups antigens incl. D,C,c,E and e

• Initially discovered in monkeys by Landsteiner and Weiner (1930’s)

• RhDantigen- important and most immunogenic- present (+), absent (-)

• 84% of europeans have RhD+

What are AB(O)H secretors

• Person that secretes antigens into bodily fluids- saliva,urine and tears etc

• 80% of caucasian population carry gene

Forensic importance of AB(O)H secretors

Blood group established from other fluids

Exclusion of non secretor

Useful historically

Red blood cells- forensic importance

• Identify blood group antigens

• Peroxidase-like activity- for detection

WBC’s- forensic importance

Contain nuclei- for DNA analysis

Plasma-

Drugs and alcohol screening

Blood pattern analysis

Serum in it- for species testing

Blood pattern analysis (BPA)

• Blood exits the body as a liquid

• Exits differently depending on injury/action- flow, drip etc

• Analysis of patterns- shows what happened/ order of events

• Violent crime- often lots of blood

Types of bloodstains- active

Blood travels by force other than gravity

• Caused by impact to body (weapon)-spatter

• Projection (punctured artery)- gushes or spurts

• Secondary object (weapon)- cast off stains

  Direction of travel on impact- tail

  Angle of impact- round vs elongated

Passive blood stains

Blood formed solely under the influence of gravity

• Blood flows often extensive- body position can affect and if body been moved post mortem

• Drops and pools- can tell time since blood shed

Transfer blood stains

Blood has been deposited and comes into direct contact with contaminated objects

• From weapon or person e.g hand or footpri t

• diposing of evidence- wiping weapon on clothes

• Indicate repeated contacts

Different resumptive tests for blood

Catalytic test-

Screening techniques

They determine presence

• Trace leveks

• Folowing clean up

• Test unknown stains

Screening techniques

ALS- for non visible traces e.g luminol

Theyre raoid and non-destructive

ALS is used to detect variety of fluids

Example luminol- spray reagent, non destructive so DNA is preserved and use light source- luminol is specifc to blood

Catalytic tests

• Test where you take sub sample of blood stain and apoly different reagents- colour change- positive

• Haemoglobin catalyses oxidation of reagents e.g H20

• Exampkes- Kastle Meyer test and Leuchomalachite green

• Will detect tiny amounts of blood but potential for fsalse postive- body creams can test positive

What is a postive result for Kastle meyer

Pink

What are confirmatory tests

• Absolutely identify unknown stain as blood

• Microscopic assessment of RBC’s and WBC’s

• Can do microscopic tests- for red and white blood cells

• Or do crystal tests- e.g teichman and takayama- formation of heamatin/hemochromagen- crystals

• Spectroscopy- is highly reliable- can tell whether haemoglobin derivatives are present

What are serological tests

Look at precipitin- identifies human proteins

• Does haemoglobin in blood react with ant-human heamoglobin

• Repeat tests using antiserums for other species

Further forensic analysis on blood type-resction of antisera with blood antigens

Associate/exclude individuals

Case examples for blood

Christoper vaughn- murder of wife and 3 children

Blamed his wife

Blood spatter evidence doesnt fit with story- dripping blood -disturbed stains

Transfer stains- moved his wife to look like suicide

Saliva

Common in sa, burgalrly

Can get them from bite marks, lip prints but can also be transferred to exhibits- ciggys, balaclavas

Non-invasive control sample collection

Forensic analysis- saliva

Detect drugs/ alcohol

Posining cases

Hormone levels

Species identification

Identify the accused

Nature of saliva

• Its a digestive aid- breaks down food starches

• Produced by 3 main glands- sublingual, submandibular, parotid)

• Humans produce 1-1.5L/day

• Contains both serous and mucus cells

• 99% water with a pH of 6.8-7.0

  -1% electrolytes, immunoglobulins,proteins, enzymes etc

  • Including digestive enzyme- a amylase

Function of saliva

Lubrication and cleansing of the mouth

Aids digestion

• broken up food-bolus

• solubillise dry good

• a amylase- breks down starch and salivary lipase for fat digestion

Anti microbial properties- e.g lysozyme

Maintains oral hygeine

Forensic analysis

Few well known presumptive tests

• enzymatic methods

• AlS screening

No currently used confirmatory tests specific to saliva

Presumptive tests rely on analysis of enzymes..

• Saliva ID is based on presence of amylase enzyme- phadebas test, sALIgAE, RSID

• Add sample of stain to soluble starch solution and iodine is added as reagent

• Looking for no reaction as if there is a reaction means starch is still present so hasnt been digested, means alpha amylase is not there

• Not specific for human saliva

• High false positive rate- due to a amylase being found in other fluids such as semen

Semen

• Evidence in sexual offences cases- person so clothing, swabs and also the scene so condoms, bedding etc

• Speed important for medical exam of victim- detectable up to 31h is mouth, 44h anus, 10 days vagina and 19 days cervix

• Aim is to identify/exclude suspect

Nature of semen

Complex fluid and cellular mixture produced by male reproductive glands

Nature of semen 1 - cellular mixture

Cellular mixture is spermatozoa cells - these are reproductive cells that carry genetic material for sexual reproduction

Nature of semen 2- complex fluid

This is seminal fluid- this is a nutrient rich environment whihc aims to protect sperm cells post ejaculation

S

Function of semen

• Sexual reproduction in humans/animals

• Contributes half genetic info to the offspring

• Average ejaculate between 2-5ml- 100-150 sperm cells/ml

• Alkaline pH 7.2-7.6

Forensic analysis- presumptive tests

ALS- but not all semen fluoresces

Test for seminal acid phosphatase most common- if present, AP will react with a naphythyl phosphate

Detect PSA (prostate specific antigens) e.g p3- present in seminal plasma= antibody-antigen reactions

Forensic analysis of sperm- confirmatory tests

• Microscopic ID of sperm cells- treated with christmas tree stain to visualise heads- nuclear fast red- nuclei and picroindigocarmine-tails (green)

• RS-ID semen strip tests

  -sensetive and specific

  -seminal vesicle-specific antigen- semenogelin

  - present in pre ejaculation fluid also

Case example semen

1968- roy tutil abducted raped and killed

1991- semen stains recovered and stored

1996- DNA profile extracted

1999- brian lunn field arrested for drink driving- saliva swab- dna match

Highlights importance of maintaining and re examinning sampkes

Vaginal fluids

• Not as common at crime scnes

• Important role in sexual assault cases

• Very few tests as fluid is not wekk defined- due to the composition varying due to menstrual cycle and variable hormone levels

Nature and function of vaginal fluids

• Vaginal secretions- mucus or fluid prodcued from cervical & vaginal glands

• Average production- 6g/day

• Protects from microbial/bacterial infections

• Proteins iclude lysozyme, amylase and acid phosphatase

• Shed blood cells, urea, epithelial cells

Presumptive tests

• No presumptive/ confirmatory tests routinely used

• Detect glycogenated epithelial cells with Periodic acid schiff reagent

  -stains cytoplasm magenta

  -only been postively used on people who menstruate and is destructive

• Lacid/citric acid ratio- used to test for vf and semen

Menstrual fluid

• Needed to distinguis between peripheral & menstrual blood

• Importance of context within the case- trauma, natural bleeding

• Challenging distinction to make- new tehcniques developing since 2017

New techniques to distinguish between peripheral and menstrual blood

• Immunochromatographic assays to detect degredation products of fibrinolysis (FDP’s)

• Found d-dimer was the most significant FDP subtype

• D-dimer was present in concs 200x greater in menstrual than peripheral blood

What is SERATEC-PMB

Detects haemoglobin and d-dimer- specifc to menstural blood

Quick, easy to use and preserves DNA

Forensic validation:

• Specific and sensetive (only need 3 microliters)

• No false postives or negatives

• Post mortem postive samples

Urine

• Useful in sexual assault, drug and abuse cases

• Difficukt to detect- low sensetivity of a available tests and false positive results

• Less viscous than other fluids

• Odour not localised

Nature and function of urine //

• Liquid by product of the metabolism

• Expels nitrogen rich by products- urea, uric acid and creatininie

• Average person produces 1.4L/day

• 91-96% water

• Contains organic and inorganic compnents

  Contains solids too like urea

Forensic tests- urine

• Diluted urine- limited ALS detection

• Urea- nesslers test looks at the activity of the ezyme urease. Breaks down urea, releases ammonia + co2- turns pink if ammonia present

• creatinine- jaffa test- creatinine reacts with pitric acid in alkaline medium

• Both have linited efficiency with dry stains

RSID urine test

Detects THP protein (tamm-horsfall)-most abundant protein- 50/100ml/day excreted

Specific to urine but concentration varies

test uses assay with THP-specific antibodies

in situ sampling to increase test sensitivity

blood prescence inhibits urine detection

not human specific

Sweat

• Least common bodily fluid found at scenes

• Useful in range of cases

• DNA extracted from clothing bedding etc

Nature and function of sweat

• Fluids secreted by sweat glands in skin

• Means of thermoregulation

• Sweat rates up to 2-4L/hr

• Mostly water with dissolved mineral traces such as lactic acids, salts and organic compounds found in urea

Forensic tests - sweat

• Most difficult bodily fluids to identify

  1- SEM-EDX- chlorine peak basis of comparison

  2- G81 monoclonal antibody’

  3-metabolite biometrics- single analyte enzymatic assays distniguish persons

Proteomics

Protein analysis- advances within bodily fluid

Proteomes- proteins coded in genetic makeup of individual (genome)

-study of proteins in a cell

-changes with environmental conditions

May yield detailed profile of an unknown individual