Body Fluids Testing

• Body fluid detection methods


  • VISUAL
  • Alternate light sources
  • Stain characteristics
  • May not be readily visible
  • MICROSCOPIC
  • Low and high magnification
  • CHEMICAL
  • Screening versus spot tests
  • Presumptive and confirmatory tests
• Relevance of body fluids? DNA profiling is not enough!
• Body fluid identification


  Provides context to the deposition of biological material
• Body fluid identification


  • Consider issues of transfer and persistence
  • Within the framework of case information
• Blood


  • Complex liquid/suspension
  • Non-newtonian visco-elastic fluid
  • 55% fluid plasma and 45% cellular components
• Plasma contains


  • 90% water
  • 8% plasma proteins (albumin, globulin, fibrinogen)
  • 1% salts
  • 0.5% food substances
  • 0.5% waste products of metabolism, enzymes, hormones and antibodies
• Cellular Components


  • Red blood cells
  • Non granular leukocytes
  • Granular leukocytes
  • Platelets
• Red blood cells


  Gas transport (O2 and CO2)
• Non granular leukocytes


  Antibody formation
• Granular leukocytes


  Immune response
• Platelets


  Blood clotting
• Kastle Meyer (KM) / Leucomalachite Green (LMG)


  Detects peroxidase activity in blood
• KM / LMG

  2 reagents: Hydrogen Peroxide and a Reduced Dye
• Die used in KM is Phenolthalein

  Leucomalachite Green (LMG) is also another test used for blood
• KM / LMG distinguish blood but not specifically human blood
• KM / LMG are highly sensitive (a drop of blood in a bucket of water)
• KM / LMG - how they work

  1. Peroxidase acts on the H2O2 releasing oxygen

  2. This causes oxidation of the dye, indicated by a colour change:

  3. Pink (KM)

  4. Green (LMG)

  5. If it looks like blood and is KM+ve/LMG+ve it is blood
• The Chemistry behind the KM test

  1. 2 stage test:

  2. Reduced KM/LMG = colourless

  3. Blood + reduced KM/LMG = colourless

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

  5. Blood + KM /LMG reagent + H2O2 -> H2O + free oxygen radical -> colour change (magenta/green)
• Hemastix® Test

  • Tetramethylbenzidine (TMB)
  • Used in solution
  • Portable - ideal for scene use
  • Positive indicated by a blue-green colour
  • Reagent strips originally designed for use in testing blood in urine
  • They are plastic strips with the test reagents bound to a pad at one end.
  • + from orange to green
• Luminol

  • Chemiluminescent - uses the peroxidase activity of the haem group in haemoglobin to catalyse the production of a blue-white light, which is short lived
  • Enhances traces of blood and is highly sensitive
  • More intense in older/decomposing blood stains
  • Needs to be in darkness
  • Glow fades quickly but can be captured with a photograph
  • Can allow pattern analysis if photographed
• Laboratory use

  1. Spot test - visible stains with a blood-like appearance

  2. Screening test - dark coloured items where blood is not readily visible

  3. Direct test - dilute stains
• • Some plant material - in particular horseradish
  • Oxidising agents such as cleaning chemicals
  • Give a reaction before the addition of hydrogen peroxide
• • Animal materials with contaminating traces of blood
  • If dye from a clothing exhibit is an issue, swap between KM & LMG
• Human blood kits - ABA Haematrace

  • Confirmatory test for blood
  • Only humans, higher primates and ferret
  • Used in instances where get a good KM reaction but not a good DNA profile
  • Cross reactivity with other body fluids - faeces, nasal secretions where blood may be present
• Semen

  • Average ejaculate: 3 ml of semen - less than a teaspoon and contains approx. 100 million spermatozoa per ml
  • 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
• Sperm morphology

  • Presence of sperm considered confirmatory for semen
  • Presence of tails may indicate recent deposition - usually lost within first 48 hours (AP activity lost between 48 - 72 hours on swabs)
• Spermatozoa are the smallest cells in the body with a total length of approx. 0.05mm
• • Middle piece and tail break off when sperm enter the egg
  • Therefore male mtDNA does not make it into cells of embryo
  • mtDNA inherited maternally
• • 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 - enzyme constituents

  • Acid Phosphatase (richest known source)
  • Alkaline phosphatase
  • Nucleotidases
  • Pyrophophatases
  • Several ATP ases
• Seminal fluid - non-enzyme constituents

  • Phosphorylcholine (only in fresh ejaculate) – immediately dephosphoryalised by AP to Choline (Choline test)
  • Hormones
  • Fructose
  • Citric acid
  • Potassium
  • Flavins
• Detection of semen

  • Can work with both wet and dry stains
  • Visual: Dry – white and crusty, Liquid – opaque, highly viscose solution
  • Crimescope – semen stains can fluoresce
  • Presumptive chemical tests
  • Microscopic examination and identification of sperm; DNA profiling of semen
• AP Test

  • 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 Test

  1. Reagent is Sodium α-naphthyl phosphate and Brentamine fast blue

  2. Acid phosphatase causes hydrolysis of α-naphthyl phosphate to sodium phosphate and α-naphthol

  3. α-naphthol reacts with Brentamine fast blue to give a PURPLE AZO DYE

  4. Strength of AP reaction characterised by: Speed of reaction, Intensity of colour change, Shade of colour (often subjective - with experience)

  5. AP breaks down 48 - 72 hours after ejaculation intimate swabs
• The Chemistry behind the AP test

  Sodium α - napthyl phosphate + AP (from semen) -> Sodium phosphate + napthol + brentamine fast blue -> Purple Azo dye
• AP screening test

  1. The AP test is a wet test:

  2. Moisten exhibit & position blotting paper

  3. Outline item and apply firm pressure

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

  5. Time reaction - initially 2 minutes...

  6. Protect item with polythene & return test paper to locate stain
• • Vaginal secretions also contain AP - although reactions tend to be pinker rather than purple and if bacteria – blue/grey
  • Many other false positives: 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


  • AP test locates stain
  • Stain extraction and microscopy of cell pellet identifies sperm
  • Assessment of amount of sperm uses an 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 (Haemotoxylin & Eosin) stain


  • Haematoxylin – stains nucleic acids purplish blue (nuclei, ribosomes and rough endoplasmic reticulum) e.g. sperm heads
  • Eosin – stains cytoplasm pink e.g. sperm tails, epithelial cells