Seminar: Detection of Semen & Seminal Fluid Stains

By :  Dr. Imra n Sa bri

Junior Resident,

De pa rtme nt of Fore ns i c Me di ci ne and Tox ic ol ogy ,

J N Me di ca l Col le ge , AMU, Ali ga rh.

      A s t he n u mb e r of ca se s of Rap e a re in crea sin g d a y b y da y th e re is a ne ed of

d et e ct io n of sp e rm a nd se me n even if p re se nt in sma ll qu an t it y, n o ma t te r h o w old th e
st a in s a re, we ha ve t o d et e ct t he se st a in s pre cise ly. Fo r d et e ct io n of sp e rm a nd
se men a re t he mo st re lia b le ma rke r fo r th e in ve st iga t ion in ca se s of rap e , sod o my,
B e st ia lit y, se xu a l mu rd e r e tc. de te ct io n o f sp e rm is a n imp o rta n t f a cto r in co n f irmin g
se xu a l a ssa u lt s. Th is pa pe r p ro vide s a b rie f re vie w a nd kn o who w o f th e n ee d f o r
d et e ct io n of sp e rms a nd se me n an d va rio u s me t ho d s a pp lie d fo r t he de te ct ion wit h
spe cia l e mph a sis on th e cu rre n t ch e mica l te st s with a ccu ra t e p ro ce d u re s f o r
p e rfo rmin g th e se t e st s.

Introduction

      S e me n is a hu ma n b od y f lu id p re se nt in hu man ma le s. I t is a viscid mu cilag in ou s

f lu id wit h f a in t ye llo w co lou r a nd cha ra cte rist ic od ou r ca lled se mina l o do u r.

      V o lu me is ab ou t 3 ml p e r eja cu la t e.

      No . o f S pe rma t o zo a = 5 0, 00 0 to 3, 50 , 00 0 p e r ml. (10 % of S P ).

      I t is a su spe n sion o f spe rma t o zoa in se min a l p la sma .  

 
 

Fi gures 1 -4 S how i ng Morphol ogy of Huma n S pe rm

S e me n co n sist of th e f o llo win g

1 . S pe rma t o zo a (10 %)

2 . S e mina l P la sma (9 0 %)

3 . E p ith e lia l Ce ll (< 1 %)

Th e S pe rma t o zo a is p ro du ced in th e t e st is by th e pro ce ss o f sp e rma to ge n e sis.

S pe rma t o zo a co n ta in s lip id, p rot e in s like p ro ta min e & h isto ne e t c. a nd en zyme s like
d eh yd rog en ise s & t ra n sa min a se s.

Th e t ot a l len g th o f spe rma t o zo a is ab ou t 50 Micro n s. it co n sist of he ad an d t a il. Th e

h ea d is f la t, o va l sh ap ed - 4 .6 X 2 .6 X 1 .6 Micro n s. in L en gt h Wid th Th ickn e ss. Th e
n ucle u s, wh ich occu p ie s ma jo r po rt io n o f t he h ea d. The ta il po rt ion is re sp o n sib le f o r
t he mo ve me n t o f sp e rm.

Th e se min a l pla sma is a mixt u re of se cre t ion d e rive d f ro m th e ma le acce sso ry

re p rod u ct ive o rga n s like e p id id ymis, se min a l ve sicle s, th e pro sta t e, va sa - de fe ra nt ia ,
b u lb ou re t h ra l & u ret h ra l g la nd s. The se min a l p la sma con t a in s Cit ric A cid , A sco rb ic
A cid , L act ic A cid, Fru ct o se , po ta ssiu m Cho lin e Ph o sp ha t e , P ro te a se s, f ree A mino
A cid s, E rgo t h ion ie ne , Zin c, Ca lciu m, Sp e rmin e , L ip id s, E nzyme s like Fib rino g en a se ,
Dia st a se , Acid & A lka lin e P ho sph a ta se , G lysid a se s, a & ß Ma n no sid a se s a & ß
G lu co sid a se s, ß G ivco u rid a se s.
ME DI CO L EG A L S IG NI FI CA NCE O F DE TE CTI O N O F SP E RM & SE ME N

Ra pe , So do my (A na l in t e rcou rse ), Be st ia lit y (Se xua l in t e rcou rse by a h u man be ing

wit h a lo we r an ima l like d og s, ca lve s, she e p e t c. ), I n ca se of fa lse Accu sa t io n b y a
wo me n , In ce st (S e xu a l in te rco u rse in b lo o d re la t ion ) a nd Se xua l Mu rd e rs.

Wh e re to lo o k fo r S e mina l S ta in s

1 . Clo t he s : Un d e rwea r, B ed sh e et , Ca rpe t , To we l, P illo w co ve r.

2 . B od y :P e rin eu m, t h ig h , V ag in a & pu b ic ha ir.

3 . S ee n of crime : O n t he f loo r o r g ra ss e t c.

Me t ho d o f Co lle ct io n

Ha nd lin g of a rt icle s b ea rin g st a in s sh ou ld be do ne ve ry ca re fu lly to avo id d a ma g e t o

spe rma t o zoa .

V ag in a l / a na l / p en ile swab s sho u ld b e sen t a lo n g wit h th e ir sme a rs on slide s. S wa b s

sho u ld be ta ke n on st e rile g au ze / clot h an d t he ir sme a rs p rep a red on ste rile slid e s.
Th e se sh o u ld b e d ried in a ir at roo m t e mpe ra tu re (3 7 d eg re e s ce lciu s) a nd swa b s
d ispa t che d in ste rile te st t ub e an d slid e s in cle an wra pp e rs.

Me t ho d s A pp lie d fo r De te ct ion o f S e mina l S ta in s a re Cla ssif ie d a s:

1 . P h ysica l E xa mina t io n

2 . Ch e mica l E xa min a t ion

3 . Micro sco p ic E xa min a t ion

P h ysica l E xa mina t io n: I n clu d e V isua l E xa mina t io n. To na ke d eye se min a l sta in s

g en e ra lly ap pe a r t ra n slu ce n t or o pa qu e sp ot s, at t ime s with ye llo wish t in t an d d a rke r
b o rd e r d ep en d in g on co lo u r an d t h ickn e ss of su b st rat a , wh ich , if a b so rb en t , a lso
a cq u ire st iff n e ss d ue to d ried se men . On go od su b st rat a se min a l sta in s ma y ap pe a r to
b e f luo re scen t un de r u lt ra vio let lig ht .

Ch e mica l exa min a t io n :

P H = 7 .4 A lka lin e .

Th e t e st s u se d to de te ct Se min a l St a in s a re :

1 . Flo re n ce Te st

2 . B a rbe rio Te st

3 . A cid Ph osp ha t a se Te st

4 . L DM I soe n zyme Me t ho d
5 . A cid Ph osp ha t a se I so en zyme Te st

6 . Cre a t in ine in P ho sph o kina se

7 . A mmo n iu m Mo lybd at e Te st (P ho sph o ru s)

8 . S e men Sp e cif ic G lyco p ro te in (P3 0 ) Te st

9 . E n zyme -lin ked immu n o so rb en t assa y (EL I SA ), t he SE MA ® assa y, f o r a se mina l

ve sicle -spe cif ic a nt ig en (S VS A )

Flo re n ce Te st

B a sis: Ch o line is d et e ct e d in th is me th o d.

P ro ce d u re: A f e w d ro p s of wat e ry so lu t io n o f t he sta in is e xt ra ct ed an d t a ke n on a

slid e an d a dro p o f Flo re n ce re ag en t (8 %) W/ V so lu t io n of Io d ine in wa t e r co n ta in in g
5 % W/V o f P ot a ssiu m Io d id e ) is p ou red & allo we d t o mix slo wly un de r a co ve r slip .
Da rk b ro wn cryst a ls of cho lin e pe rio d id e , g en e ra lly ne ed le sh a pe d , f o rmed wit h a f e w
min u te s. Non - sp e cif ics & f a lse n eg at ive re su lt s a re co mmo n.

B e rb e rio ’s Te st:

B a sis: De te ct io n o f S pe rmin e

P ro ce d u re: A f e w d ro p s of Be rbe rio ’s rea ge n t wh en a dd ed to sp e rma t ic f lu id p rod u ce s

cryst a ls of spe rm in p icra te (n ee d le sha p ed , rho mb ic & of ye llo w co lou r).

Fo r va riou s va lid re a son s, like no n -sp e cif icit y an d la ck of rep ro d u cib ilit y, t he f lo ren ce
a nd be rbe rio ’s te st s h a ve no t be en accep t ed u n ive rsa lly.

A CI D P HOS P HA TAS E SP O T TE S T:

Mo d if ied Fish man a nd Le rne r’s met ho d . Th e f lu id ob ta in ed af t e r th o rou gh ma ce ra t io n

o f a sma ll clot h p ie ce (a b ou t 4 mm2 ) is p la ce d in a ca vit y on a p o rce la in t ile la nd t wo
d rop s e ach of cit rat e bu ff e r (P h 4 .9 ) a nd 1% W/ V aq ue ou s so lu t io n o f d isod iu m P he n yl
p ho sph a te a re a dd ed . Af te r 1 0 min u te s t he ph en o l is de te ct ed by t he ad d it io n of 2
d rop s o f p he no l re ag en t & 2 d ro p s o f 20 % W/ V so lu t io n o f so d iu m ca rb on at e . Blu e
co lou r d e ve lo pe d wh ich ind ica te s t he p re se n ce of a cid p ho sp h at a se.

L DH I SO E NZY ME ME THO D

De te ct io n o f S pe rma t o zo a

P ro ce d u re: S e min a l sta in s a re e xt ra ct ed wit h 1 ml o f wa t e r. 0 .2 5 ml o f cle a r ext ra ct is

mixe d wit h 0 .2 5 ml of 4 0% W/ V o f su cro se. 0 .1 ml o f t h is mixtu re is sub je cte d to
ve rt ica l po lya cryl a mide g e l e le ct ro p ho re sis. E le ct ro ph o re sis is ca rrie d o ut in
re f rig e rat o rs f o r 1 50 min ut e s usin g a cu rre n t of 5 m A Iso en zyme ba nd s a re re vea le d
b y st a in ing .

Th is me th od g ive s a sp e cif ic b io che mica l de te ct ion of spe rma t o zo a in se men in th e

p re se n ce o f V ag t ina l Flu id , B lo od , Na sa l S ecre t io n, S a liva & Urin e.
A CI D P HOS P HA TAS E IS O E NZY ME ME THO D

P ro ce d u re: S e mina l st a in s a re e xt ra ct e d wit h wa te r a nd is used in po lya crylmile ge l

E le ct rp h o ret ic me t ho d f o llo wed b y st a in in g wit h me t h yl be llif e ryl. Ph o sp ha t e re a ge n t
e na b le t he se min a l a cid p ho sph a ta se to be dist in gu ish ed f ro m th at o f o th e r su b sta n ce
like vag in a l se cre t ion s.

Th e me t ho d is su f f icien t ly spe cif ic & ap p lica b le to se me n d e rived f ro m no rma l,

o lig o sp e rmic a zoo spe rmic & va se ct e mize d in d ividu a ls.

A d va nt a ge s:

1 . L DH iso en zyme is sta b le in sta in s fo r o ve r 4 wee k.

2 . I soe n zyme pa tt e rn o f hu man is d iff e ren t f ro m th at o f a n ima l.

3 . P o sit ive re su lt s a re ob ta in in la rg e n u mbe r o f ca se s.

4 . Ca n dif fe re n t iat e f ro m va g in a l se cre t io n s o n p at t e rn o f b an d s.

CRE A TI NI NE P HO S P HOK I NA SE :

B a se s: De t e ct io n of crea t in ine ph o sp ho kin a se . No rma l se mina l f lu id co nt e nt - 3 85 -

1 40 00 U of CP K/ W.

Dia g no sis: >40 0 V o f CP K / ml.

A d v: E n zyme is st ab le & can b e de mo n st ra t ed in old st a tu s o f six mo nt h s.

CHO L I NE AND S PE RMI NE TE S T:

B a se s: Un iq u e co mb ina t ion o f cho lin e & spe rmin e is p re se n t o n ly in se men .

L iqu id se me n & d ried se min a l st a in s ca n b e id en t if ied b y a th in la ye r ch ro mat o g rap h ic

Te chn iq ue .

1 ML o f se men p re se n t ca n b e d et e ct e d b y th is me th od .

MI CRO S CO P I C EX A MI NA TIO N:

Th e Micro Scop ic d et e ct io n of th e Se min a l st a in s is b a se d in mo rp ho lo g y o f

spe rma t o zoa .

Micro sco p ic de te ct io n o f sp e rma to zoa . Clo th p ie ce s f ro m dif fe re n t st a in s a re ta ken in

0 .5 ml o f 0 .0 1 N HCL in sma ll t e st t ub e s p la ce d in a be ake r con ta in in g wa t e r. A f te r
son ica t ion f o r 5 min ut e s th e e xt ra ct s a nd th e clo t h p ie ce s a re t ra n sf e rre d t o se pa ra t e
micro sco pe slid e s a nd clo th p ie ce s de lica te ly t ea sed wit h a ne ed le . Th re ad s a re
re mo ved a nd th e re sid ua l liq u id is g en t ly eva po ra te d to d ryn e ss. Re sid ue o bt a in e d is
st a in e d h ae ma to xylin a nd eo sin.

Flu o re sce n ce Micro S co p y:

I s a lso u se d f o r de te ct io n o f sp e rma to zoa . It is b ased o n t he p rin cip le th a t Y Ch ro mo
so me is f lu o re sce n t t o q u in a crin e. Wit h th is me t ho d it is p ossib le to de te ct bo th in ta ct
spe rma t o zoa a s we ll a s th e d iscon n e ct ed h ea d s.

Co n clu sio n

Wit h t he ab ove re vie w it is q u it e clea r t ha t a lt ho u gh th e re are so ma n y t e st a va ila b le

f o r th e d et e ct io n of se me n & spe rms req u ire d t o e xa mine in ca se s of rap e, se xu a l
mu rd e r, un na tu ra l, se xu a l of fe n ce s. The se te st s h a ve th e ir wo n limit a t io n s ad va n ta ge s
a nd d isa d va n ta ge s. Fo r va rio u s va lid re a so n s like n on - sp e cif icit y an d la ck of
re p rod u cib ilit y th e Flo re n ce a nd be rb e rio ’s te st s ha ve n ot be en acce pt ed u n ive rsa lly
a pa rt f ro m po sit ive Flo re n ce t est can a lso b e ob ta in ed f ro m ot he r bo dy t issue
con t a in in g ch o lin e. Ne ga t ive Flo re n ce ma y be ob ta in ed f ro m se min a l sta in in ca se th e
cho lin e co n te n t is lo w o r in ca se s wh e re th e st a in s a re d e co mp o se d . Ho we ve r fa lse
p osit ive t est a re n ot ob ta in ed in ca se s of va g ina l se cret io n .

A cid ph osph a ta se of p ro st at ic o rig in is a lso ut ilize d fo r t he b io - ch e mica l d et e ct io n o f

se men . An y int e rf e ren ce of se ru m a cid p ho sph a ta se ca u se d by th e ad mixt u re o f b loo d
in th e se min a l sta in s wa s t ill re ce nt ly ru le d ou t b y ta king - a dvan t ag e of th e f a ct, t ha t
o n ly p ro sta t ic a cid p ho sph at a se is in h ib ite d by 1 -ta rt ra te . Th is dist in ct ion , ho we ve r,
b eca me in va lid af t e r th e d e mo n st rat io n by Willo t t in 19 72 an d Da vie s an d Wilso n in
1 97 4, t ha t va g ina l A cid p ho sph a ta se co u ld a lso b e in h ib ite d by 1 -ta rt ra te . Ap a rt f ro m
t he se te st s o th e r te st s like Ra d io -immu n o assa y an d co mme rcia l e nzyme -lin ke d
immu n o so rb e nt a ssa y (E LI S A ) a re a lso a va ila b le . Co mme rcia l en zyme -lin ked
immu n o so rb e nt a ssa y (E LI S A ), th e S E MA® a ssa y, fo r a se min a l ve sicle - sp e cif ic
a nt ig en (S VS A ) p ro vid e s h ig h ly se n sit ive d et e ct ion of se me n bu t t he se te st s are co st ly
e no ug h re st rict in g th e ir u se fo r re sea rch p u rp o se o nly.

Th e o n ly me t ho d s a mo ng t he ava ila b le o ne s wh ich a pp ea r t o b e con sid e red as spe cif ic

a nd un a mb ig uo u s, are th e micro sco p ic o r e le ct rph o re t ic de te ct ion o f spe rma t o zoa in
se min a l st a in s de rived f ro m no rma l in d ividu a ls a nd th e e le ct rp ho re t ic d et e ct io n of
se min a l Acid ph o sp ha t a se in st a in s orig in at in g f ro m a ll in d ividu a ls whe t he r n o rma l o r
a bn o rma l.