EFFECT OF PHYSİCOCHEMİCAL FACTORS ON STABİLİTY OF ABH ANTİGENS.

Effeet

o

f

Physicoehemieal

Faetors on Stability of

ABH

A

n

tigens

D.V.

D.V.K.

V.K. KASHYAP

Central rorensic Science Laboratory, I3ureau of Police Research & Development,

Ministry of Home ACfairs, Ramanthapur, Hyderabad - India

FİzİKOKİıvIYASAL FAKTÖRLERİN ABH ANTİJENLERİNİN STABİLİTESİ ÜZERİNE ETKİsİ

Özet

Kan lekelerinin en uygun muhafaza ve laboratuvarlara gönderim şeklini belirlemek üzere, 5 aylık süre içerisinde farklı çevre koşullannda tutulan lekelerdeki AI3H antijenlerinin dayanıklılığı incelendi. AI3H tiplemesi için rutin olarak kullanılan 3 kan grubu yönteminden yararlanııaL I3unlar absorpsiyon-elüsyon,

kanşık aglutinasyon ve absorpsiyon-inhibisyon yöntemlcriydi. Aşırı iklim koşullarının ABH tiplemesinin

doğru bir şekilde yapılmasını engellediği görüldü. Düşük nem oranının ve nötral pH değerlerinin muhafaza için en uygun ortamı oluşturduğu gözlendi. Absorpsiyon-elüsyon yönteminin diğer tekniklerden daha duyarlı olduğu saptandL

Summary

The stability of AI3H antigens in blood stains exposed to different environmental eonditions up to a period of five months, was studied to identify appropriate conditions for storage and transportation of stains to

[orensic laboratory. Three routincly used blood grouping teehniques were used to type ABII substances in stains afıcr their exposure ıo di[[erent test conditions. Extreme elimatic condiıions wcre found to have and

ad verse a[[ecl on AI3H substances thereby a[[ecting their detection by all the studied methods. Low

ıemperature, low or no humidiıy and neutral pH were ideal conditions for sıoring the stains till ıhey received

by crime laboratory for examination. Absorpıion cluıion teehnigue was round more sensitive and provided cornpatiblc results among all the meıhods followed in ıhe sıudy.

Kcy words : J\l3lJ antigens - Blood stains - Blood grouping -Environmental jactors - Antigen stability

146 D.V. RAO, D.V.K. RAJU,V.K. KASHYAP

INTRODUCTION

Since

t

he d

i

sc

o

very of A

BH

sys

t

e

m

by Landsteiner

(1)

i

n 1900, consi

d

erable

w

or

k

has

been done to eluc

i

da

t

e the na

t

ure of the

system

and

it

s ap

p

li

cati

o

n

s

i

n

F

o

r

ensic

Science.

ABH

ty

p

ing

of [

r

es

h

b

l

o

o

d is

si

m

p

le

but in

s

ta

ins w

h

erei

n t

h

e

r

ed

are

ruptured,

it

can't

b

e grouped

by

simp

le

agglutinati

o

n meth

o

d

(2).

Specia

l

agglutination

methods have to be

ado

p

ted

for

group

in

g blood

stains

.

Various

p

rocedures for

t

y

pi

ng

ABH antigens in

stai

ns

have

been repof

ted o

f

wh

i

ch

mixe

d

agg

l

ut

in

a

tio

n

, a

b

sorp

t

ion

elution

and absorpt

i

o

n

in

h

ibitio

n

are

si

m

p

l

e,

sensiti

ye an

d

best

esta

b

lishe

d

meth

o

ds

which

are

routinel

y

pr

acticed in

f

ore

n

s

i

c

scie

n

ce

labora

t

ory

(3,4). Recent

l

y,

th

e

app

li

cation

of

immuno

a

ssay

techn

i

ques for t

h

e detec

ti

o

n

of

b

l

ood

grou

p

a

n

tigens

i

n

stains

has be

en

widely

repo

rt

ed (5-7)

.

B

u

t th

ese

metho

d

s

n

eed specia

l

skills and

equipment

and wo

u

Jd ne ed

tim

e

t

o perc

o

l

a

te

i

nt

o

t

he fo

r

ensic

practice. Aging a

n

d

starage co

n

diti

o

n

o

f

stai

n

s

are

c

r

itical factors in success

f

ul typi

n

g of

sta

in

s. Bac

t

eria

l

contaminati

o

n

and

p

u

trifaction o

f t

he

stains

give

false pos

i

ti

v

e

reac

ti

ons

. Ex

po

sure

to

extrcme

climatic

co

n

dit

ions may e

au

se

de

n

a

tur

ation

o

f

a

n

tigens,

he

n

ce poar rate of

succcss

in

ty

p

i

n

g.

A

d

e

t

a

i

le

d

s

t

udy o

f

the

effec

t o

f

vario

us

env

i

ro

n

me

ntal

e

o

ndit

i

ons

on

t

he

stab

i

li

t

y of

ABH antigens

in

stai

n

s a

n

d

also their detectability by

routine

m

ethods

i

s

very

mu

c

h

needed

d

ur

i

ng

stcps

t

o ensure

success

f

ul

detection

. I

n

t

his

e

n

deavo

ur

,

b

l

ood stains

exposed t

o

differe

n

t

en

vi

ron

m

ental

c

o

n

ditions

f

or a period of

5 mont

h

s, are ty

p

ed

by

three

prevalent

metho

d

s to

evalua

t

e

the

sta

bi

1ity of ABH

m

o

l

ecules

an

d

a

l

so

t

o

find

out

a most

su

itable techniq

u

e to

d

etermi

ne

AB

H

s

u

b

stanees

i

n

forensic ex

h

i

b

its

.

MATERIALS AND METHODS

Chemicaf: All the chemicals and reagents used in the study were of analytical grade and procured from Sigma Chemicals Co. (USA).

Anıisem: Anti-A, Anti-B, Anti-H were purchased from Orthodiagnostics (New Delhi).

lllood Slains: Fifty blood stains on cotton doth were prepared from the blood obıained by pricking different volunteers whosc blood group s wcre previously confirmed, and divided in 10 groups comprising each of 5 stains prepared from the blood of different individuals. Each group contained all the three (A, B, II) positive stains.

Physicochemica! Factors

Temperalure Condilions: Two batehes of the stains were kept in BOD incubators mainıained at 20°C and 40"C rcspectively. Third and fourth batehes of the stains were kept in two hot air ovens set at 60"C and 80"C. All the four batehes of stains were maintained at the adjusted temperature for the entire period of study.

pll Condilions: pH 3.0, 5.0, 7.0 and 9.0 werc adjusted in individual beakers conıaining acid washcd sand with suitablc cuffer. At each pH a batch of stains was kept during the studied period.

lIumidiıy: One batch of stains was maintained at 50 % and another at 100 % relative humidity in humidity chamhers.

]~vping Melhods: Mixed agglutination teehnique of Coombs and Dodd (8), absorption clution method of Iluward and Marıin (9) and absorption inhibition method of Boyd (LO) werc used for the typing of the ABII antigens. Fibres tcased from the stains were used for typing by various established mcthods.

RESULTS

AND

DISCUSSION

The

effec

t of

storing of blood

stains

at different temperatme,

relative

hu

mid

ity

an

d

pH

on

th

e det

ectability

of

ABH

antigens

by

different methods are

shown

i

n Tab

l

e

I, II

and III,

r

espectivel

y.

Exposme

to

hi

gher

t

empera

tm

e (above

60'C) was

more

d

eleterio

u

s

for

t

he ABH

antigens.

The

degree

o

f

damage

increased

t

h

e

ri

se

of te

m

peratme

and

p

eriod of

exposme (F

i

gure

1

). Th

e

ABH

antigens

in

stains exposed

to

h

i

gher h

um

idity

(1

00

%)

were poorly deteeted by the

m

ethods

(Figure

2).

Both

extremes of

p

H

of

t

he

environme

nt

had signifieant damaging effect o

n the detect

abi1ity

o

f

ABH

antigens.

Optimum

results

were obtained with stains store

d

at neutra

l

pH.

Red

blood

ceU antigens are highly stable at norma

l

tempara

tm

e, hu

m

i

dity

an

d

pH,

but

their epitopes are

disorganised

severly when exposed

to

extreme co

n

ditions of these

[aetors thereby

mak

ing

their

deteetability

impraetieal. The natiye strueture of

the

blood

group

antigens

is

b

asic

erilerion

for

thei

r d

etection

b

y

their

speeific antibodies. Various

ınethods

are reported for AB

H

grouping

of

old

blood stains (11

,12).

ABH antigen typing

not only in

blood

stains but also in di

ff

erent body Duid

stai

n

s could

a

l

so be

sueeessfully

earried out

(13-

15).

The

deciine

in

the deteetabi

l

üy of ABH

antigens

in

s

t

ains stored

at

hig

h

er

temperatures

is d

u

e to

t

he

st

m

ct

m

a! as well as fun

etiona!

de

formatio

n

s

t

h

e

ant

igens

by the

higher

t

emperature (

1

6)

.

The degree

of

damage gener

a

ll

y

de

p

ends

u

po

n

temperature

and period of exposme. The percentage of suecess

ful

detection

o

f

AB

H

antigens

was higher in

stored at

20'C

temperature and

hence the

sensitiv

it

y peaks

of

all

the

m

ethods were found at this

t

emperature only (Figme 1).

Tablc 1. Effcct of temperature on ABII detectabiliıy

Dctcctability of stains by three mcthods in percentage

Tempcraıure

CC)

A B LI A B H A B ı-ı

20 100 100 100 100 100 100 100 100 100

40 60 60 40 40 40 20 40 40 20

60 20 20 O O O O O O O

80 O O O O O O O O O

The pH

of

the

h

as

p

r

ofound

in

Duenee

on the

deteetion

of

ABH

substanees

due

to

change

i

n the

ne

t

electr

i

cal eharge

over

the

m

olee

ule

wh

i

e

h

may lea

d

to

the alteration

in

t

he

antigenicity of

the

m

oleeules

and

co

n

sequentl

y

af

f

eeting

the

ir

identifiea

ti

on with antibodies. This

was

the

reason

f

or the

de

cl

i

ne

i

n

thc dc

t

cetion

of

stains stored at aeidie

and

basic pH eondirions

b

y any o

f

the meth

ods.

148 pH 3 5 7 9 Humidity 50 100 %0/ ABH posilive stains

D.V. RAO, D.V.K. RAJU,VK. KASIIYAP

Tablc 2. Effeeı of pH on ABH detectability

Deectability of stains by three methods in percentage

Absorption dution Mİxed agglutination Absorption inhihition

A B II A B H A B H

o

o

o

Tablc 3. Effeet of humidity on ABI-! dctectability

O 20 100 20 O 20 100 20

Detecıahility of stains by three methods in percentage

Ahsorption elııt ian Mİxed agglmination Absorptian inhibition

O O 100 O A B II A B LI A B II 100 40 100 80 60 40 20 80 80 100 40 20 20 80 20 80 O 100 20 Absorption inhibition Mİxed agglutination Absorption clution 10 20 30 40 50 60 70 80 90 100 'C 80 20 80 O

Figurc 1. Deteetability of ABIl antigcns in stains, exposed to different tcmperaturcs, by the studicd method S_

% of Al3l! pas it ive stains

l()() 80 60 40 20 20 40 60 80 100 Relative humidity % Absorption cJution Mixed agglutination Absorption İnhibition

Figurc 2. Erfeet of hıımidity on the dctcctability of ABII antigens in stains.

% of ABII posilive stains 100 80 60 __ 40 20 10 II 12 13 14 pll of environmenı Absorption clution Mixed agglııtination Absorption inhibition

150 D.V. RAO, D.V.K. RAJU,V.K. KASHYAP

Sensitivity peaks

f

or

a

ll

the

three

me

th

ods we

r

e not

i

ced at neutral pH

where the

external

influences

on

the net

electrical charges

of t

h

e antigens

is

rulc

d

ou

t.

Hum

i

dity

encourages microbia

l

grow

th

(17)

and

hig

h

er hum

i

d

i

ty favours t

h

e

i

r

gro

wt

h

on the

stains

.

The

secre

t

ory

substances of

th

ese

m

i

crobes

damage

antigenic

substances

of RBC

wh

i

ch

r

esult in

the poor detectab

i

l

ity of

a

ntigens i

n

sta

in

s kept

a

t

hig

h

er rela

ti

ve

hpmidity (18).

From

tbe res

u

lts

it

i

s

ev

i

dent t

h

at the

absorption

clut

i

o

n

is

more sens

iti

ye and

provides

com

p

atib

l

e

results

t

h

an the other two

methods.

This study

co

n

cludes

t

hat the

following

precautions

need

to

be

observed for s

u

ccessful detection of ABH

antigens

i

n

old stains; (1)

the

stains

mu

st

be

transported

t

o

the

laboratory

in

a mois

t

ure

h

ce and

seale

d

po

l

ythe

n

e bag s ; (2) if

the

temperature

o

f

t

he

environmen

t

is

above 20'C,

it

is

preferable

t

hat

t

he stains

i

n

polythene bags

may

b

e

kept

in ice box while

t

r

ansportalion

to avoid

exposure to

hig

h

er temperature; (3) if

the blood

stains

a

r

e

foun

d

on ac

id

or

a~kaline

surface,

it

would

be adv

i

sable,

th

a

t the sta

i

ns

may be

trans

f

erred o

n

to blotLing

paper

str.ips or steril

i

sed cotton

fi

bres.

REFERENCES

Souhradev, J.M. (1983) in Serological Methods in Forensic Science, (Rolih, S.D. and Judd, W.J.,eds)

pp. 19-21., American Assoeiation of Blood Banks, Arlington, Virginia.

2 Bhatnagar, RK. (1987) Ind. J. Forensic Sci., 1, 25-28.

3 Sivaram, S., Sivaraınan, C.A. (1979) 1. Ind. Acad. Farensic Sci., 18, 28-29. 4 Kabat, E.A. (1956) Blood Group Substances, Academic Press, New York. 5 Davie, M.J. (1980) J. Farensic Sci. Sac., 17, 267.

6 Bmner, Jr. K.W., Kiss1ing, C.W (1979) Transfossian, 19, 773.

7 Cunningham - Rund1es, Ch., Feng, Z.K. (1988) 1. Immunoı., 140, 3880.

8 Coombs, R.R.A.,Dodd, B. (1961) Med. Sci. Law, 1,359-377. 9 Howard, H.D., Marties, P.D. (1969) 1. Farensic Sci. Sac., 9, 28. 10 Boyd, W.c., Boyd, L.G. (1937) J.lmmunol., 33, 159-172. 11 NickoUş, L.D., Perira, M. (1962) Med. Sci. & Law, 3, 172-179. 12 Olltleridge, RA (1963) Nature, 198, 698.

13 Poon, W.L., Dodd, B.E. (1964) Med. Sci. & Law, 4, 258.

14 Davies, A., Lincoln, P.J., Martin, P.D. (1983) in lOth International Congress of the Society for l/aemogenetics, 77, München.

15 Ho1burn, A.M., Masters, E. (1974) Br. 1. Haematol., 28, 157.

16 Hakomori, S.I., Kobatov, A. (1974) in The Antigens, Vol. 11 ( Sela, M.,ed) p. 79, Academic Press,

NewYork.

17 Kashyap, V.K., Raju, D.V.K., Bhatia, R.Y.P. (1988) Adli Tıp Derg., 4, 121-126.

IS Davis, R. (1963) in The Biochemistry of Industrial Microorganisms (Rainbow, c., Roose, A.H., eds)

pp. 68-150, Academic Press, London.

Reprints request to:

Dr. V.K. Kashyap

Central Forensic Science Laboratory, Bureau of Police Research &

Development, Minist!), of Home AHairs, Government of India, Ramanthapur, Hyderabad - 500 013 India