Body fluid testing

visual detection methods

alternate lighting sources

stain characteristics

may not be readily visible

microscopic detection methods

low and high magnification

chemical detection methods

screening versus spot tests

presumptive and confirmatory tests

what is the relevance of detection methods?

-DNA profiling isn't enough

Body fluid identification provides context to the deposition of biological material

-consider issue of transfer and persistence

-within framework of case info

what is blood?

Complex liquid/suspension

55% plasma and 45% cellular components

DNA profile is in the white blood cells

presumptive tests for blood

• Kastle Mayer (KM)/ Leucomalacite Green (LMG)

• Detects peroxidase activity in blood

• 2/3 reagents: hydrogen peroxide and reduced dye

-phenolthalein (KM)

-Leucomalacite Green (LMG)

• Distinguish blood human and animal

• Highly sensitive more KM than LMG

how do presumptive tests for blood work?

Peroxidase acts on H2O2 releasing oxygen

Causes oxidation of the dye indicated by colour change

 pink (KM) green (LMG)

Destructive technique

chemistry of Kastle Mayer test

2 stage test:

Reduced KM/LMG = colourless

Blood and reduced KM/LMG = colourless

Blood and reduced KM/LMG and [O] = coloured (intense magenta/blue/green)

KM is explosive so CSI cannot carry equipment to use it

chemical equation for what occurs with the blood presumptive tests

Blood + KM /LMG reagent + H202 H20 + free oxygen radical colour change (magenta/green)

Hemastix test

Tetramethylbenzidine (TMB)

Used in solution

Portable- ideal for scene use

Positive indicated by blue-green colour

Reagent strips originally designed for use in testing blood in urine

Plastic strips with test reagents bound to a pad at one end

+ from orange to green

luminol

Chemiluminescent - uses peroxidase activity of haem group in haemoglobin (Fe2+) to catalyse the production of a blue-white light

Mainly made of water and is done at end as it is destructive and has to be done in pitch black

Enhances traces of blood and is sensitive

More intense in older/decomposing blood stains

Glow fades quickly but can be captured with a photo

Can allow pattern analysis is photographed

More sensitive= increase in false positive anything containing iron will test positive

Chemical doesn’t inhibit/ruin samples

Can be used on cold cases as it is so sensitive able to detect but not necessarily get a profile from it

Bleach is main false positive

lab use of presumptive test

• spot test-visible stains with a blood-like appearance

Rub stain with folded filter paper, unfold, add KM/LMG then H2O2

• Screening test-dark coloured items where blood is not readily available

Methodically rub sections of the item with folded filter paper and test each as above

• Direct test-dilute stains

Cut thread from exhibit and test directly on filter paper

false positives for blood presumptive tests

• Some plant material- in particular horseradish

• Oxidising agents such as cleaning chemicals

Give reaction before addition of h2o2

• Animal materials with contamination traces of blood

• If dye from clothing exhibit is an issue, swap between KM and LMG

 

human blood kits - ABA Haematrace

• Confirmatory test for blood

• Only humans, higher primates and ferret positive result

• Like covid test

• Used in instances where get a good KM reaction but not a good DNA profile

• Cross reactivity with other bodily fluids -faeces, nasal secretions where blood may be present

 

menstrual blood test

Like covid test

Looks for d-dimer as marker for menstrual blood

semen

3ml of semen per ejaculation

Slightly alkaline pH of 7.2-7.7

Testes produce 200-300 million sperm every day

Pre-ejaculate can be both AP and sperm rich

More sperm found in 1st portion of ejaculate

Can be variable e.g. repeated intercourse, decreases volume

Long abstinence can increase volume up to 13ml 100 million spermatosa per ml

Need 6 to produce profile

Whole semen is composed of cells so can get DNA profile from vasectomised males

sperm morphology

Presence of sperm considered confirmatory for semen

Presence of tails may indicate recent deposition-usually lost within first 48 hrs

Cell wall is highly resistant to anything

what happens to sperm tails?

Spermatosa are the smallest cells in body with a total length of approx., 005mm

• Middle piece and tail break off when sperm enter egg

• Therefore male mtDNA doesn't make it into cells of embryo

• mtDNA inherited maternally

sperm tails

• Not often seen in casework, consequence of slide preparation as much as natural degradation

• Vaginal swabs- tails lost usually within 8-10hrs of ejaculation

• Anal swabs- less likely to find tails on sperm compared with vaginal swabs

• If tails are present- very recent ejaculate

seminal fluid

 rich in enzymes and non-enzyme constituents

seminal fluid - enzyme constituents

 acid phosphatase (richest known source)

• Alkaline phosphatase

• Nucleotidases

• Pyrophosphatases

• Several ATP-ases

seminal fluid - non-enzyme constituents

• Phosphorylcholine (only in fresh ejaculate)- immediately dephosphorylated by AP to choline (choline test)

• Hormones

• Fructose

• Citric acid

• Potassium

• Flavins

detection of semen

works with wet and dry

visual detection of semen

• Dry-white and crusty

• Liquid- opaque, highly viscose solution

• Crime scope- semen stains can fluoresce

chemical detection of semen using presumptive tests

• AP reagent

• Florence iodine (choline test)

microscopic detection of semen

Microscopic examination and identification of sperm; DNA profiling of semen

AP

acid phosphatase

presumptive tests for semen

• AP test- high levels in seminal fluid

• Detects the presence of Acid Phosphatase in seminal fluid

• Acid Phosphatase is water soluble, therefore washing removes AP activity

• Its presence is independent of the presence of spermatozoa

• Semen stains are often not visible- AP test used to screen fabric items (to locate areas for further testing)

• Or to test supernatant from a swab extraction

AP (acid phosphatase test)

• Reagent is sodium a-naphthyl phosphate and brentamine fast blue

• Acid phosphatase causes hydrolysis of a-naphthyl phosphate to sodium phosphate and a-naphthol

• a-naphthol reacts with brentamine fast blue to give a purple azo dye

• Strength of AP reaction characterised by:

• Speed of reaction

• Intensity of colour change

• Shade of colour (often subjective-with experience)

• AP breaks down 48-72 hours after ejaculation intimate swabs

chemical reaction for the AP test

Sodium a-naphthyl phosphate + AP (from semen)

 

 

Sodium phosphate + naphthol + brentamine fast blue

 

 

Purple Azo dye

AP screening test

• Wet test

• Moisten exhibit and position blotting paper

• Outline item and apply firm pressure

• Remove blotting paper & spray in fume cupboard with a chemical reagent used to detect AP

• Time reaction - initially 2 mins

• Protect item with polythene and return test paper to locate stain

limitations of AP test

• Vaginal secretions also contain AP- although reactions tend to be pinker rather than purple and if bacteria- blue/grey

false positives for AP test

• Cauliflower/sprouts - pink/brown

• Tea - strong purple

• Some toiletries - strong purple

• Spermicides - grey reactions

• Faeces and urine - purple

removal of cellular material

• Sodium dodecyl sulfate (SDS)

-detergent

-denatures membrane proteins & unfolds them into polypeptide chains

• Proteinase K (ProK)

-an endopeptidase, breaks polypeptide chains into smaller molecules

-named for keratin hydrolysing properties

• Sperm remain intact, tails are lost (but often fall off during swab/stain extraction anyway

microscopy - confirmatory test for sperm

• AP test locates stain

• Stain extraction and microscopy of cell pellet identifies sperm

• Assessment of amount of sperm uses and arbitrary numbering system (trace, 1+ to 4+), linked to persistence data

• Heavy cell coverage can be problematic- use of SDS & Proteinase K to remove them

H&E (haematoxylin and eosin) stain

Haematoxylin- stains nucleic acids purplish blue (nuclei, ribosomes and RER) e.g. sperm heads

Eosin- stains cytoplasm pink e.g. sperm tails, epithelial cells

Florence iodine (choline test)

Identifies presence of choline found in high level in seminal fluid

Choline is water soluble- not detected after washing

Not as sensitive as AP and cannot be used for screening

Short golden-brown crystals indicate a positive result

christmas tree stain

Nuclear fast red & picroindigocarmine (PIC)

Sperm heads (nucleus)

Epithelial (cytoplasm) cells green

saliva

Watery fluid secreted from salivary glands of mouth

Humans produce 1-1.5 litres per day

what does saliva consist of?

• Mucin

• Alkaline phosphate

• Thiocyanite ions

• Nitrate ions

• Cells- mainly bacteria, but also epithelial and leukocytes

• Salivary amylase

what is saliva and what does it help do?

It is an alkaline homogenous fluid

Aids breakdown of food for easy swallowing

Initiates digestion of starch using amylase

Buccal epithelial cells- not a true component of saliva but become mixed as comes from mouth lining

detecting saliva

Look for visual stain

Screening/testing for activity of enzymes normally present in saliva (Phadebas test/amylase paper)

Looking for buccal epithelial cells

amylase

-enzyme catalyst - alpha and beta forms

Chromosome 1 has 2 loci coding for amylase:

• AMY1

Saliva, breast milk, perspiration

Concentration 50x greater in saliva

• AMY2

Pancreas, semen, vaginal secretions

Phadebas test

Made of starch polymer linked to blue dye marker- insoluble in water

Amylase breaks down starch and releases blue dye

Paper sprayed with Phadebas solution or in test tube add tablets to water and supernatant

Apply paper to moistened item and press for 5-10-40 mins

Tube test is incubated in 37 degrees for 30 mins

Amylase breaks down starch releasing the water soluble blue dye

Presumptive test- detects alpha amylase

Azure blue dye attached to starch complex sprayed onto paper

Spray with iodine working solution

limitations of Phadebas test

Time consuming and requires regular observation

Poor sensitivity- false negatives

Poor specificity:

• Vaginal amylase is problematic in sexual offense investigations

• Faecal amylase is indistinguishable- although faecal staining Is visible

Buccal cells

• Buccal cells are squamous nucleated epithelial cells

• Similar to those found in moist orifices such as the vagina

• NOT a confirmatory test

• Can help ID a suitable sample for DNA

• May assist in interpretation that saliva is present

urine

Watery solution of metabolic wastes such as urea, creatinine dissolved salts and organic materials

Ph close to neutral

Not considered good source of DNA

Colour varies but more identified by distinctive smell on stained materials (ammonia, breakdown product of urea by bacteria)

May see a tide mark

No screening tests so need to know where you expect urine to be

Selected areas can be tested using creatinine and urea tests

Creatinine test

Creatinine provides energy for muscle contraction, is unstable and is present in serum, RBC, sweat, bile and gastrointestinal fluids

No function in the body and is a waste material which is filtered through the kidneys, concentrated in the tubules and then excreted in urine

Concentration of creatinine varies dependant on concentration of urine

Produces orange colour with picric acid in alkaline medium

Colour depends on concentration of creatinine which is dependent on diet/liquid intake

urea test - DMAC

Nitrogen wate- metabolite of protein

Initial product of protein metabolism is ammonia which is toxic to body

Mammals excrete ammonia in form of urea dissolved in urine

Urea produced by liver is filtered and excreted in urine-small amount excreted in sweat

DMAC test- +stains will turn magenta

faeces

End product of digestion

Consists of:

Undigested/indigestible food

Bacteria

Mucus cells

Average 100g daily

Visible brown stain due to bilirubin presence

Odour due to breakdown of amino acids into skatole and indole by bacteria

testing for faeces

Small amount of stain can be diluted in water, spotted onto slide and examined microscopically

Things may be seen:

• Seeds

• Starch grains

• Parasites

• Bacteria

• Yeasts

• Hair

urobilinogen test

Bile pigment excreted in faeces, which fluoresces with the addition of a reagent

Produces a 2 phase solution

View under low wave UV light

Orange, yellow or apple green fluorescence in lower phase indicates urobilinogen

Variation in colour is diet dependent

faeces and DNA

Generally seen on items as smears- sensitivity increased with the introduction of DNA-17

High bacterial content so degradation of DNA

sweat

Distinct odour

Stains white/tide mark

Test

-extract

-prepare slide

-examine for epithelial cells

No real detection tests

 

vomit

No detection tests

Distinct smell

Can be tested for

-amylase

-acids (Ph)

-microscopic food stuffs

skin tissue

Impacted skin can be found on items

Identify presence of possible skin under LPM-can be examined under HPM if critical to case

Report as 'apparent skin'

Important in assault cases where IP has been subject to blunt force trauma e.g. on weapons or on shoes following kicking/stamping assault

Examination strategies should include looking for impacted skin on such items

blood

presumptive

-Kastle Mayer

-Leucomalacite green

-luminol

-haemastix

confirmatory

-ABA haematrace (human specific)

-microscopy (H&E)

semen

presumptive

-AP test (acid phosphatase)

confirmatory

-microscopy (H&E, Christmas tree stain)

azoospermic

no sperm count

oligozoospermic

low sperm count

saliva

presumptive

-Phadebas

—no specific confirmatory test but can do microscopy to identify epithelial/buccal cells

KM reaction

Testing for haemoglobin (has peroxidase activity)

Peroxidase splits h2o2 reagent into h2 and o2

[o] and phenolphthalein causes pink colour change

Pretty much instantaneous result

False positives:

• Horseradish

• Rusty items

• Cleaning products (oxidising agents)

Phadebas reaction

Testing for amylase in saliva

Amylase hydrolyses starch causing a blue-dye to be released

Phadebas solution

Detects alpha amylase

Azure blue dye attached to starch complex sprayed onto paper

Spray paper with iodine working solution

AP reaction

Testing for acid phosphatase

Reagents: sodium a-naphthyl phosphate + brentamine fast blue

Purple colour = +ve

Acid phosphatase hydrolyses a-naphthyl phosphate into sodium phosphate and a-naphthyl

a-naphthyl reacts with brentamine fast blue- purple colour

blood false positives

Plant materials e.g. horseradish

Oxidising agents- cleaning products

semen false positives

Tea-purple

Cauliflower/sprouts-pinky/brown

Batteries-grey

Vaginal secretions-pink