Semen, Saliva, and Other Fluids

spermatozoa

confirmation of the presence of semen; ~10-25% of semen composition
stored in the epididmys where udnergo functional maturation
transfered through vas defferens to ejaculatory duct, into urethrra whre joined wwith seceretuibns

seminal fluid

secretions from seminal glands

sperm head

contains nucleus with densely packed chromosomes 5×3 micrometers

midpiece

short nec containing mitochondria provides energy for moving tail

tail

sperm motility

accesory gland secretions ~75-90

prostate 30 and seminal vescicles 60

prostate

PSA and acid phosphatase

seminal vescicles

secreted proteins play role in coagulation of the ejaculate; contains flavin

flavin

causes semen to fluorescenc

typical ejaculation

1-6 mL, average 3
meatabolic activity of glands, time from previous ejacualte, paticual ductal

Normospermia

~50-150 millional/mL

Oligospermia/aspermia

oligo = < 20 million/mL
aspermia = no sperm
may be transient due to illness, nutricional deficiency, therapeuting drug usechronic cases: congential deformities, chemotherapuetic drugs, exposure to industrial cheicals, vasectomy

SAP Test

phosphorlcholine (PC) synthesiszed in seminal vescicles
choline formed by

Presumptive/screening tests

Polyamines - spermine (Barberio crystal test), choline (florence crystal test)
Enzymes: SAP (seminal acid phosphatase)

Barberio test

spermine synthesized in prostate - enhancement of sperm motility
spermine phsphate prsumptive crystal test react spermine with anions (phosphate or picrate)

Florence test

Phosphorylcholine (PC) synthesized in seminal vescicles
choline formed by hydrolysis of PC by SAP (SAP originates in the prostate)
occurs subsequent to mixing of these secretions
insoluble choline periodide crystals when treated with a solution of iodide and potassium iodine (KI and I)

SAP seminal acid phosphatase presumptive test

present in high levels of semen, 100-1000 fold greater than otjher body fluids, synthesized in the protestae
most common screening test
enzyme which cleaves a variety of organic phosphates
elevated levels fo SAP in blood indicative of prostate cancer

Seminal acid phosphatase

active at acid pH, not affected by vasectomis, sensitive bt not specific
also vaginal acid phosphatse but slower reaction than SAP for some substrates

PSA/p30/KLK3

major protein in seminal fluid: 300-4200 ug/ml, ~1200 ug/ml
high molecular weight of 30 kDA, respoinsible for hydrolyzing semenogelin
       semenogelin creates gel matrix around ejaculated sperm, then after p30/PSA action, gel is broken down allowing sperm to move more freely 

PSA confirmatory?

present in urine and peripheral blood - low concentration
small quantities in fecal material, sweat, and breast milk

P30 ID methods

crossover electrophoresis
ouchterlony double diffusion
immunchromatography
4ng/ml sensitivity (ie 1 in 10^6 dilution)
can detect p30 in mae

ELISA

P30 ELISA

antibody-antigen-antibody (Ab-Ag-Ab) sandwich ELISA
monoclonal antibody Anti PSA → Rabbit polyclonal antibody anti → swine anti rabbit IgG Alkaline Phosphatate conjugate

Sperm ID - confirmatory

normal semesn contains 96% abnormal forms of sperm, 1-2% immature
other cellular components: immagture germinal cell types, leucocytes, epithelial cells

abnormal space

large head, small head, double tail, double head, tapered head (anal beads), coiled tail, immature form (egg looking), shapeless head (amorphous), cytoplasm dropless (still have cytoplasm), abnormal midpiece

process for confirmatory sperm

extract form subsstrate: physical removal by agitation, destruction of substrae by acid/base digesion, observation while remaining attached to thread
visualized: directly without staining, use of histological stains

Christmas Tree Stain

nuclear fast (NF) red/picroindigocarmine solution (PICS)
sperm heads pink/red, everything else green

density of sperm under microscope

++++many in every flield
+++many in some or most fields, easy to find
++some in some fields, easy to find
+hard to find
few, very small number on whole sldie

0 none

sperm - human vs animal

rabbit, ram, boar, bull, other primates: same length/ovoid shape as humans
mouse and rat: hook shaped head

Stability of semen constituents

dried stains on clothing/bedding etc: SAP, p30, sperm stable for months of years, lost by washing
post-coital samples(living victim vaginal swabs): variation in persistence of SAP (12-48 hrs), p30 (1-3 days), sperm (3d-5d); tails ~24 hours, persist in cervix for longer periods of times

post-coital sampels (deceased): often persist until putrefaction

semen lost form vaginal environment dye to

drainage, dilution with vaginal fluid, phagocytosis of spermatazoa by netrophilic lymphocytes and mononuclear cells

Evidence collection

evidentiary items collected upon consent of victim
consider: assault activity, time elapsed since the assault, post asault (showering, abthing, brushing teeth, mouth washing, vomiting, douching, bathroom)
age and gender
guided by background histroy produce including the suspects actions during the assualt, if not full range of samples

recommended collection time frames

vaginal - 120
anal - 72
oral - 24
bitemarks - 96 hours 

Saliva

1-1.5 L of saliva produced
largely water - contains small quantities of electrolytes, proteins, antibodies, enzymes, oral epithelial cells
produced from salivary glands - 70 submandibular glands (neck), 25 parotid(back of mouth) glands, 5 sublingual glands(under tongue)
continuous basal level is maintained - increased during eating, digestive aid

presumptive tests available saliva no confirmatory

inorganic anions (thocyanate and nitrate), enzymes (alkaline phosphatase or a-amylase)

Saliva

a-amylase: hydrolyzes a 1-4 bonds in starch cannot hydrolyze branched alpha 1-6 bonds
AMY1 and AMY2 highly homologous, antibodies recognize both

AMY1

SALIVARY locus; perspiration, breast milk

AMY2

PANCT|REATIC locus; semen urine fecal material vaginal

Amylose

straight linear chain of glucose units a 1-4 glycosidic

Amylopectin

branched polymer, containing a 1-6 linkages at branch points

debranching enzyme

hydrolyzes 1-6 linkages at branch points, then a-amylase can break down straight 1-4 chains

Saliva-starch/iodine reaction

amylose forms helices: 6 glucose residues per turn, <12 glucose/2 helical turns = no color change

Saliva Assays pretri dish radial

radial diffusion
agar and starch in well, add sample
diffusion of a-amylase in saliva (sample diffusion)
add iodine: turn blue/purple outside, if saliva present then CLEAR ring with diameter proportional to enzyme concentration

Differentiate saliva assays

AMY1 - inhibit salivary with wheat lectin
AMY2 - inhibit pancreatic with kidney bean extract

Phadebas Test (Cibacron Blue-Starch) Dye stach substrates saliva assay

blue starch polymer, dye covalent attached to starch. a-amylase will release dye into solution. colorimetric anaylsis (620)
amylase present will break down starch and dye releases
positive: BLUE, negative: CLEAR

SALIgAE kit

commercially available, simple and sensitive, more accurate than other
changes to yellowunknown mechanism, proprietarty

Immunochromatography RSID

RSID saliva, commercially available
Anti-human salivary a-amylase (monoclonals). one conjugated to collodal gold (in conjugate pad in sample window), other immobilized at Test zone
Control zone: immobile anti IgG
two red lines positive

sensitive- as little as 1 microliter
cross reacts with vaginal secretions, fecal amterial, and breast milk

Urine

useful in sexual assaults and assaults involving urination
homicides involving ligature or manual strangulaion (involntraility urinate prior to death)

Aqueous solution- largely consisting of water
urea is the most abundant waste product: from elimination fo ammonia produced from metabolic process of amino acids
inorganic: Cl, PO4, SO4
organic: creatine,creatinine, urea, urinay indican, urochrome, purine/pyrimidines, steroid derivatives
excrete
excrete 9-29ml/kg/day
~55-70 g solids

urinary system

eliminates solubled toxic wastes that are cellular metabolic by products
kidneys, ureter, bladder, urethra
formaion of urine takes place in the kidneys

filtered to reabrosb water, glucose, nutrients
waste (ions ammonia, urea,, etc) secreteed through ureter to bladder before its excreted through the urethra

Identification of Urine

Odor: gentle heating and odor detection - odor resulting from ammonia that is released from the breakdown of urea by bacteria
Inorganic ions: SO4-2 - barium salt, PO4-3 - ammonium phosphomolybdate (yellow), Cl -, AGNO3 (AgCl precipitate)

DMAC Urine Presumptive testing

0.05% para dimethyl amino cinnamaldehyde
detects amines (particular urea) , add acetone/HCl, if urine present turn dark pink/red

Creatinine (Jaffe)

add picric acid w/base to creatinine = creatinine picrate (bright red), but picric acid explosive when dried

Tamm Horsfall protein )THP)

most abundant protiein in urine, ~40% of proteins
exclusively synthesized in epitherlial cells of Henle’s loop (Kidney)
biological function not fully understoof
ELISA or Immunochromatographic (RSID-Urine) using Anti-THP antibodies
NOT CONFIRMARTORY bc urine diffiuses (dries)
ELISA more sensitive, no reported cross reactivity with other body fluids

Vaginal Secretions

no tests available for US labs

Menstrual blood

no tests to differentiare from peripheral blood

Fecal material

Macroscopic exam (color, odor)
Microscoping (undigested foodstuff)
Testing-detection of urobilinoids
Heme degradation products that are excreted into fecal material
Add zinc acetate in ethanol solution → green fluorescence in UV