Long-term storage of blood prior to whole-blood lymphocyte culturing

Veterina~ Immunology and Immunopathology, 2 (1981) 555--560 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

LONG-TERM

J.M.B.

STORAGE

KANEENE

I

OF BLOOD P R I O R TO W H O L E - B L O O D

, F. S O P E R

2

, D.W.

JOHNSON

2

LYMPHOCYTE

and R.K.

555

CULTURING

ANDERSON

2

Icollege of V e t e r i n a r y Medicine, M i c h i g a n State University, E a s t Lansing, MI 48824, U.S.A. 2 D e p a r t m e n t of Large A n i m a l C l i n i c a l Sciences, college of V e t e r i n a r y Medicine, U ~ i v e r s i t y of Minnesota, St. Paul, M N 55108, U.S.A. (Accepted

21 S e p t e m b e r

1981)

ABSTRACT Kaneene, J.M.B., Soper, F., Johnson, D.W. and Anderson, R.K., 1981. storage of blood p r i o r to w h o l e - b l o o d l y m p h o c y t e culturing. Vet. I m m u n o p a t h o l . , 2: 555--560. Studies term blood

storage

conducted

culture

to i n v e s t i g a t e

cattle was utilized.

A a n d B, f o l l o w i n g mediu~

was added

collection.

(A) was

The cultures

the k e e p i n g

application

quality

of these

of b l o o d

findings

were

divided

left intact w h i l e

following

collection.

incubated

It was o b s e r v e d

thereafter.

into

RPMI-1640 Both

and a p o r t i o n and a s s a y e d that

Addition

for 7 days prior

of long-

Peripheral

was

for a total of 7 days

into their DNA.

g o o d for 4 days but d e t e r i o r a t e d

B prolonged possible

day.

incorporation

culturing.

from each animal

Part A was

w e r e t h e n kept at r o o m t e m p e r a t u r e

thymidine

lymphocyte

Blood

to p a r t B i m m e d i a t e l y

e a c h b l o o d was t e s t e d e v e r y [3H]

the effect on b l a s t o g e n e s i s

of b l o o d prior to w h o l e - b l o o d

f r o m normal

2 parts,

parts

were

Long-term I~nunol.

of

for

i n t a c t blood

of RPMI-1640

to culturing.

to

The

are discussed.

INTRODUCTION

A whole-blood study

(Kaneene

several

lymphocyte

et al.,

advantages

stimulation

1978a).

o v e r the pure

(WBLS)

It was p o i n t e d lymphocyte

a s s a y was

reported

in a p r e v i o u s

out that the W B L S a s s a y h a d

(PLS)

a s s a y in that

it was simpler,

0165-2427/81/0000--0000/$02.75 © 1981 Elsevier Scientific Publishing Company

556 faster and less costly. of

In another

study,

s e n s i t i v i t y and s p e c i f i c i t y and were

attributes

assay. dies

no c o n v i n c i n g our

reports

to s u p p o r t

l a b o r a t o r y from d i f f e r e n t

involve

long hours,

b l o o d prior I) w h e t h e r

further the p r a c t i c a l

that belief.

This

and 2) w h e t h e r

then

lymphocyte

We,

however,

blood samples

to d e t e r m i n e

study,

addition

Since

stu-

found

are sent to

and since s h i p p i n g of these samples may

felt e s s e n t i a l

to WBLS culture.

Since

in terms

use of the WBLS

for in vitro

therefore,

the

was

blood could be held for a long time and still

W B L S assay, diately

it was

1978b).

f o l l o w i n g collection.

states

compared

in terms of these 2

(Kaneene et al.,

It has been g e n e r a l l y b e l i e v e d that blood

s h o u l d be used w i t h i n 48 hours

were

found c o m p a r a b l e

( s e n s i t i v i t y and specificity)

we have been e n c o u r a g e d to i n v e s t i g a t e

the 2 a s s a y s

of a c u l t u r e

l o n g - t e r m s t o r a g e of

designed

to d e t e r m i n e :

be good for use in

m e d i u m to the blood imme-

f o l l o w i n g c o l l e c t i o n w o u l d i m p r o v e the k e e p i n g q u a l i t y of blood

for the

assay.

MATERIALS

AND M E T H O D S

Animals

Twenty-flve normal this

study.

Science,

and h e a l t h y cows of 2 to 21/2 y e a r s of age were

T h e s e a n i m a l s were

from a herd at the D e p a r t m e n t

utilized

in

of A n i m a l

U n i v e r s i t y of M i n n e s o t a .

Bleeding and testing schedule

E a c h animal was b l e d and t e s t e d 3 times

C o l l e c t i o n of b l o o d and e x p e r i m e n t a l

in a one month period.

design

A p p r o x i m a t e l y 40 ml of b l o o d were c o l l e c t e d by j u g u l a r v e n i p u n c t u r e e a c h a n i m a l and p l a c e d into one s t e r i l e tube c o n t a i n i n g heparin. I r e a c h i n g the laboratory, p l a c e d into 2 s t e r i l e

b l o o d was d i v i d e d into equal portions;

tubes.

B l o o d in one tube was

c o v e r e d and kept at room t e m p e r a t u r e was d i l u t e d 2-fold, mM).

perature was

s u p p l e m e n t s were added.

until the end of the study.

for 7 days,

c o l l e c t e d was

20 ml each,

and

left intact and p r o p e r l y

B l o o d in the r e m a i n i n g tube

using RPMI-16402 culture medium

No serum or other

temperature

until used.

from

After

containing L-glutamine

(2

Both tubes were kept at room

B l o o d s a m p l e s were kept at room tem-

t e s t i n g p o r t i o n s of each blood e v e r y day.

i d e n t i f i e d as day i.

I 50 U/ml; The U p j o h n Company, K a l a m a z o o , M i c h i g a n 2 G r a n d I s l a n d B i o l o g i c a l Company, G r a n d Island, N e w York

The day blood

557 Preparation

of b l o o d c u l t u r e s

Blood culture (Kaneene

Culture

was c o n d u c t e d

as r e p o r t e d

in our earlier

study

1978a).

conditions

Concanavalin were

preparation

et al.,

cultured

A 3 was

u s e d at a c o n c e n t r a t i o n

for 6 days.

t h y m i d i n e 4 labeling, as p r e v i o u s l y

harvesting,

described

s t u d y were a n a l y z e d

The rest of culture

of 1.0

and s c i n t i l l a t i o n

(Kaneene

et al.,

u s i n g a student's

/culture.

conditions,

1978a).

All cultures

[3HI

spectrometry The data

counting

generated

were

in the

t-test.

RESULTS

It was o b s e r v e d culturing

yielded

(Figure

i) that blood without

a significant

ConA-induced

added R P M I - 1 6 4 0

response

Without RPMI (n=25)

(P<0.001)

prior

to

up to 4 days.

With RPMI (n=25)

I o ×

3

OU I

f Control

/Control 0

,

i

i

1

2

3

,

,

4

5

6

7

1

2

3

4

5

6

i 7

0

H o l d i n g T i m e (Doys)

Fig. I. W h o l e - b l o o d l y m p h o c y t e s t i m u l a t i o n responses i n d u c e d by C o n c a n a v a l i n A in blood samples kept for various days at room t e m p e r a t u r e prior to culturing. V e r t i c a l bars r e p r e s e n t s t a n d a r d errors of the means. 3 ConA; M i l e s - Y e d a , Rehovot, Israel 4 S c h w a r z / M a n n , Orangeburg, N e w Y o r k

558 A f t e r 5 days,

the C o n A - i n d u c e d r e s p o n s e d e c l i n e d and was not s i g n i f i c a n t l y dif-

f e r e n t from c o n t r o l

cultures

a d d e d prior to culturing,

(P>0.5).

c a n t l y high even up to day 7.

The c o n t r o l c u l t u r e s

The difference between ConA-induced were

significant

In blood samples where

the C o n A - i n d u c e d r e s p o n s e s

(P<0.001)

responses

lymphocyte

RPMI-1640 was i) were

signifi-

had a fairly good response.

and r e s p o n s e s

for all the 7 days.

t h a t b l o o d for w h o l e - b l o o d

(Figure

In general,

s t i m u l a t i o n test was

in c o n t r o l

good even after

h a v i n g been kept for 4 days at room t e m p e r a t u r e p r i o r to culturing. c u l t u r e m e d i u m of RPMI-1640

cultures

it was o b s e r v e d

to the b l o o d f o l l o w i n g its collection,

Adding a prolonged

the k e e p i n g q u a l i t y of b l o o d up to 7 days prior to culturing.

DISCUSSION

Numerous cyte

papers

have been r e p o r t e d d e s c r i b i n g the u s e f u l n e s s

s t i m u l a t i o n test in v a r i o u s

1978c,

Senogles

Karkowa

et al.,

and Wallace,

W o o d w a r d et al., (PLS)

assay.

tists

in the early

lymphocyte 1972,

1979,

1979)

diseases

1978, T a r n o v i c Rossie

(Faber et al.,

and Holm,

et al.,

1979,

Due eo time and cost

Pauly et al.,

(WBLS)

1972,

lymphocyte

1973,

(Kaneene et al.,

Ayivor

1979,

1979, stimulation scien-

(Paty and Hughes,

et al.,

comparable

1978b).

s u r r o u n d i n g the use of the l y m p h o c y t e

et al.,

the p o s s i b i l i t y of w h o l e - b l o o d

a s s a y in d i f f e r e n t d i s e a s e s

P a u l y et al,

s e n s i t i v i t y and s p e c i f i c i t y

Kaneene

i n v o l v e d in r u n n i n g the PLS assay,

1970's s t a r t e d to i n v e s t i g a t e

stimulation

1978,

K a n e e n e et al.,

S o l l o d and Frank,

u s i n g the t r a d i t i o n a l p u r i f i e d

r e p o r t e d that both the PLS and WBLS a s s a y s were

concern

1978,

of the lympho-

1976).

R e c e n t l y we

in terms of their

However,

the f u n d a m e n t a l

s t i m u l a t i o n assay is p r e s e r v a t i o n

of the b l o o d s a m p l e well and long e n o u g h to be t e s t e d when the l a b o r a t o r y concerned

is ready.

In an e a r l i e r study

a short p e r i o d at v a r i o u s temperature,

(Senogles et al.,

temperatures

as o p p o s e d to f r e e z e r or o r d i n a r y

y i e l d e d the m o s t s i g n i f i c a n t b l a s t o g e n e s l s . that blood perature

for WBLS a s s a y was

1978) we kept blood for

and o b s e r v e d that blood kept at room refrigerator

In the p r e s e n t

temperatures, study,

we o b s e r v e d

good even after h a v i n g been kept at room tem-

for 4 days prior to culturing.

A d d i n g a culture m e d i u m of RPMI-1640

to the b l o o d f o l l o w i n g its c o l l e c t i o n p r o l o n g e d the k e e p i n g q u a l i t y of blood up to 7 days prior to culturing. We

s p e c u l a t e that blood samples,

in q u a l i t y

(Figure

where

no c u l t u r e m e d i u m was added,

I) due to d i m i n i s h e d n u t r i e n t s

declined

and i n c r e a s e d m e t a b o l i t e s .

A d d i n g the c u l t u r e medit~n f o l l o w i n g c o l l e c t i o n of blood p r o v i d e d a d d i t i o n a l nutrients

to the cells and a d i l u e n t to m e t a b o l i t e s .

cial to add a c u l t u r e m e d i u m to blood s a m p l e s tion.

This w o u l d be p a r t i c u l a r l y useful

Thus,

it may be b e n e f i -

i m m e d i a t e l y f o l l o w i n g its collec-

if samples are e x p e c t e d

a l a b o r a t o r y far from the place of collection.

Secondly,

to be sent to

it m a y also be useful

559 in cases where n o t ready with

b l o o d was

to be c o l l e c t e d

to test it right

the culture

medium

away.

taken

applications

as r e c o m m e n d a t i o n s

for a d d i t i o n a l

studies

Lastly,

but the l a b o r a t o r y

it may be useful

in the tube prior

tory then will h a v e a choice ~he possible

from a patient,

to c o l l e c t i o n

is

to mix the c o a g u l a n t

of blood.

The

labora-

to run the b l o o d that day or later. of our o b s e r v a t i o n s

by all means.

a r o u n d this

mentioned

T h e y are intended

fundamental

above

should

not be

to serve as stimuli

area.

ACKNOWLEDGEMENTS

This

research

Experiment Service,

was

Station

U.S.

supported

by grants

of V e t e r i n a r y

Department

from the U n i v e r s i t y

Services,

Animal

and Plant

of M i n n e s o t a Health

Inspection

of Agriculture.

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