A rapid microtechnique for in vitro stimulation of canine lymphocytes using whole blood

Veterinary Immunology and lmmuno pathology , 1 (1980) 251--261 Elsevier Scientific Publishing Company, Amsterdam -- Printed in Belgium

251

A RAPID MICROTECHNIQUE FOR IN VITRO STIMULATION OF CANINE LYMPHOCYTES USING WHOLE BLOOD

P.J. FELSBURG, M.T. REILLEY, and J.K. SINNIGEN Clinical Immunology Laboratory, Department of Clinical S t u d i e s , School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

(U.S.A.)

(Accepted 22 November 1979)

ABSTRACT Felsburg, P.J., Reilley, M.T. and Sinnigen, J.K., 1980. A rapid microtechnique for in vitro stimulation of canine lymphocytes using whole blood. Vet. Immunol. Immunopathol., i: 251-261.

A simple, rapid microtechnique utilizing whole blood has been developed for evaluating the in vitro stimulation of canine lymphocytes. The test uses 5 ul. of heparinized whole blood per culture in a microtiter plate and requires no serum supplement. Cultures are harvested by an automated multiple sample cell harvester. This technique permits large numbers of replicate samples to be tested on individual animals with minimal amounts of blood and minimal technician time.

INTRODUCTION The lymphocyte transformation test is a widely accepted in vitro correlate of the cell-mediated immune system in clinical and experimental immunology.

The in vitro response of peripheral blood

lymphocytes to mitogenic stimulation has become a standard test in most clinical immunology laboratories for evaluating the cellmediated immune competence of patients with various immunologic disorders.

Conventional lymphocyte transformation tests are based

upon the separation of lymphocytes from whole blood and performing the test either in culture tubes or micro culture plates Oppenheim and Schecter, 1976).

(reviewed in

Unfortunately, these procedures, in

addition to being laborious and time-consuming, require relatively large volumes of blood.

This factor is an important limitation in

veterinary medicine when one is evaluating the immune competence of young puppies or small breed dogs.

Lymphocyte transformation tests

0165--2427/80/0000--0000/$02.25© 1980Els~ierScientificPubli~mgCompany

252

using whole blood have h e l p e d to a l l e v i a t e this p r o b l e m 1970;

Han and Pauly,

1972; Park and Good,

A l t h o u g h these tests require c o n s i d e r a b l y

(Junge et al.,

1972; Gerber and Brown,

1974)

less blood than the

conventional

techniques,

they still present c o n s i d e r a b l e t e c h n i c a l

difficulties

in h a r v e s t i n g of the cultures,

many tests at any p a r t i c u l a r time.

especially when performing

Recently,

several m i c r o t e c h n i q u e s

e m p l o y i n g whole blood have been r e p o r t e d w h i c h have e l i m i n a t e d the technical difficulties Razzano, et al.,

in the h a r v e s t i n g p r o c e d u r e

1973; Eskoly et al.,

1975; Lassila et al.,

(Kaplan and 1976 Shifrine

1978).

The purpose of this paper is to d e s c r i b e a m i c r o c u l t u r e w h o l e blood l y m p h o c y t e t r a n s f o r m a t i o n test in the dog w h i c h permits the e v a l u a t i o n of the in vitro r e s p o n s i v e n e s s of p e r i p h e r a l blood lymphocytes to optimal and s u b o p t i m a l c o n c e n t r a t i o n s of PHA and P ~ using as little as one ml. of blood.

M A T E R I A L S AND M E T H O D S

Do@s Eight p u r e b r e d beagles and 12 other p u r e b r e d or m i x e d breed dogs w e r e used for this study.

The beagles w e r e part of a normal dog

colony and the other dogs were m a i n t a i n e d as blood donor dogs at the U n i v e r s i t y of P e n n s y l v a n i a Small A n i m a l V e t e r i n a r y Hospital.

All dogs

were y o u n g adults and in good h e a l t h at the time of the study. Both males and females were r e p r e s e n t e d in the study group. Data from 8 to 16 w e e k old puppies were o b t a i n e d during b a s e l i n e e v a l u a t i o n prior to various e x p e r i m e n t a l procedures.

Blood Blood was c o l l e c t e d by v e n i p u n c t u r e into syringes c o n t a i n i n g preservative-free heparin

(20 u n i t s / m l of blood).

The blood was

i m m e d i a t e l y t r a n s f e r r e d into sterile plastic culture tubes.

Culture medium R P M I - 1 6 4 0 culture m e d i u m supplemented with streptomycin,

(Flow L a b o r a t o r i e s ,

i00 u n i t s / m l of penicillin,

Rockville, MD)

100 ~g/ml

and 2 mM g l u t a m i n e was used in all experiments.

Mito@ens Phytohemagglutinin-P

(PHA-P; Difco Laboratories,

MI; one lot) and p o k e w e e k m i t o g e n

Detroit,

(P~4; Grand Island B i o l o g i c a l Co.,

253

Grand Island, NY; one lot) were r e c o n s t i t u t e d with sterile saline, a l i q u o t e d in small volumes, experiment,

and stored at -70°C until use.

For each

aliquots w e r e thawed and d i l u t e d to the d e s i r e d

c o n c e n t r a t i o n s in RPMI-1640.

The c o n c e n t r a t i o n s r e f e r r e d to in thiE

study are ug. per ml. of culture medium.

Culture C o n d i t i o n s Whole blood lymphocyte cultures w e r e set up in sterile m i c r o t i t e r tissue culture plates w i t h U - s h a p e d bottoms Linbro S c i e n t i f i c Co., Hamden, CT).

(Linbro IS-MRC-96TC,

V a r i o u s volumes of u n d i l u t e d

whole blood were added to each well w i t h E p p e n d o r f m i c r o l i t e r pipettes using sterile tips.

RPMI-1640,

with or w i t h o u t mitogens,

was added to the wells in v a r y i n g volumes by the same method. Control cultures c o n s i s t e d of whole b l o o d d i l u t e d in R P M I - 1 6 4 0 alone. All tests w e r e p e r f o r m e d in t r i p l i c a t e and i n c l u b a t e d at 37°C in a humid,

5% CO 2 a t m o s p h e r e for v a r y i n g i n c u b a t i o n p e r i o d s .

experiments,

In several

the cultures were incubated at 39°C,

M e a s u r e m e n t of DNA synthesis D u r i n g the last 16 hours of incubation, tions of 3 H - t h y m i d i n e

(2.0 Ci/mmole;

5 pl. of v a r y i n g concentra

New England Nuclear,

Boston, MA)

diluted in m e d i u m was added to each well.

H a r v e s t i n g of cultures After inclubation,

the m i c r o c u l t u r e s w e r e h a r v e s t e d onto glass

filter strips by an a u t o m a t e d cell h a r v e s t e r R e s e a r c h Institute,

Rockville, MD).

e x t e n s i v e l y w i t h d i s t i l l e d water,

(Model 24V, B i o m e d i c a l

The cultures w e r e w a s h e d

3% acetic acid, and again w i t h

d i s t i l l e d water in order to lyse the red blood cells. drying,

After air

the filter discs were placed in s c i n t i l l a t i o n vials,

s c i n t i l l a t i o n in toluene L i q u i f l u o r

(New England Nuclear,

and

Boston, MA)

was m e a s u r e d in a P a c k a r d Tri-Carb C2425 liquid s c i n t i l l a t i o n spectrometer

(Packard Instruments,

Downer's Grove,

are p r e s e n t e d as the net counts per minute or s t i m u l a t i o n ratio

IL).

The results

(CPM) per volume of blood

(S.R.), i.e., the ratio of CPM in the p r e s e n c e

of m i t o g e n s divided by the CPM w i t h o u t mitogens. RESULTS A pilot e x p e r i m e n t was c o n d u c t e d in order to d e t e r m i n e certain optimal conditions

for a micro w h o l e blood lymphocyte t r a n s f o r m a t i o n

test for the dog.

This study was p e r f o r m e d by d i l u t i n g h e p a r i n i z e d

254

whole

blood

RPMI-1640 into

on days

i:IO,

optimum

fourth

test

of

The

day was

a PHA dose was

of undiluted containing The

whole

medium

following

and medium

medium

Serum

the

approached and

were

problem

dogs.

mitogen

us

The

cultures

with

performing

needed

if o n e w a s

this

reason,

to w e l l s

volume already

concentrations. volumes

used

were

All

provided based.

the

For

directly

200 pl.

harvested

a specified

s e t up u s i n g

of b l o o d

mitogens.

study was

in w h i c h

added

were

experiment

of t u b e s

the d i l u t i o n s

i0 pl.

and without

otherwise

a test

containing

aliquoting

and harvesting

on several

the desired

and

cultures

from this

The major

could be

tubes

of w h o l e

blood

in the p i l o t

added

cultures

to were

iOO a n d

2 0 0 ~i.

harvested

effect dog

of

on day

indicated.

supplementing

serum

cultures

is i l l u s t r a t e d

with

O%

0"25'10" 5'

the

in F i g u r e

serum

suppressed

5%

JO%

0'28"10" §'

cultures

the

I.

with

5% a n d

10% p o o l e d

In g e n e r a l ,

response

to PHA.

supplementing This was

0 '~t" I0" 5 "

P H A - P ( / ~ / ml )

Fig.

of

supplement

The normal

with

Five

with

4 unless

response

to d e v e l o p blood

The

the number

experiments

which

experiment.

was

of PHA,

the p r e s e n t

1:20,

optimum.

in t h i s m a n n e r

made

results

blood was

an a t t e m p t

in c u l t u r e

plate.

upon which

conducting

1:40

concentrations

5.

information dilution

on the

and

of a m i c r o t i t e r

3, 4, a n d

the

1:20,

with various

each well

with

the

1:5,

i.

Effect

of s e r u m

supplements

on the PHA response

in 4 d o g s .

255

more pronounced

in the 5 ~i. cultures.

serum has no inhibitory, ~i.

nor enhancing,

The only cultures

in which the

effect were the I0 ~i.;

i00

cultures.

Concentration

of 3H-th[midine

Due to the small volume of blood in the cultures, varying concentrations of 3H-thy midine were evaluated. Figure 2 shows the results of adding O.i, 0.25, hours prior to h a r v e s t i n g

and 0.5 pCi/well of 3H-thymidine

(day 4).

16

The optimum response appeared with

the addition of 0.5 ~Ci/well which is the standard concentration other m i c r o c u l t u r e

o.,~.,

~. ¢ 0

a

10

|

5 " 2.5"

lymphocyte

transformation

o..,. ~

o.s IAcl

in

tests.

_" IOFJ ; IOOFi o to/a ; 3ooFI

0'10'5'2.5'

'0"10"5

"?.5"

P H A - P (Fg/ml)

Fig.

2. Effect of varying concentrations response in 5 dogs.

of 3H-thymidine

on the PHA

Days of harvest Figure

3 illustrates

after 4 days incubation. diminished

that the peak stimulation with PHA occurred Cultures harvested on day 5 showed a slightly

response when compared to the response observed on day 4.

Incubation temperature Since the normal body temperature

of the dog is approximately

we compared the PHA dose response of four dogs at 37°C Over-all, cultures

(Table I).

there does not aDDear to be any advantage of incubating at 39°C.

39 °C, the

256

DAY Z --

i0 ¢

DAY 3

OAY 4

GAY 5

lO 1~ 5~1 ~ ti=0/,/,I

----SF! ; 200~1 I0 ~u.Ii IO01,&l o io FI ; ~ooFi

0 ' I0'

5 '?.5'

0 'I0'

5

25

PHA-P

Fig.

3.

TABLE PHA

Effect

of

day

of

12~5

0 ilol

the

PHA

5 12.RI

(/zg/ml)

harvest

on

response

in

5 dogs

I

dose

response

in

cultures

incubated

at

37°C

and

37 ° PHA-P (ug/ml)

CPM

39°C. a

39 ° S.R.b

CPM

S.R.

0

45

+

4c

i0

7885

+

2504

178

+

62

10878

+

4489

179

+

80

5

92

.....

11643

+

2117

263

+

61

13389

+

4155

222

+

9260

+

1262

209

+

39

10730

+

2690

177

+ 74

1

4099

+

2750

158

+

58

9166

+

2314

146

+

31

0.5

3145

+

1960

72

+

43

5718

+

1708

89

+

18

n=4 c

9

2.5

a b

63

S.R.

=

Stimulation

mean

+

standard

Ration

deviation

257

To examine

the p o s s i b i l i t y

their peak r e s p o n s e incubated

at 39°C and h a r v e s t e d

No a c c e l e r a t e d

TABLE

that cultures

to PHA earlier,

response

incubated

cultures

on day

at 39°C may have

from four dogs w e r e

2, 3 and 4 (Table

II).

was observed.

II

PHA dose

response

according

to day of h a r v e s t

in cultures

incubated

at 39°C. a

PHA-P(ug/ml)

Day 2

Day

3

Day

39 + 5

4

0

45 + 14 b

I0

892 + 654

2227 + 1663

4985 + 3179

5

1520 + 1291

3610 + 2546

7075 + 3454

2.5

1197 + 868

3976 + 3116

8144 + 3119

1

787 + 624

3148 + 2263

5593 + 2796

0.5

472 + 260

1729 + 1087

3441 + 2273

a

a n=4 b mean

CPM + s t a n d a r d

Optimum

conditions

following

b lood

for the tes~

conditions

is added

concentrations

_

deviation

On the basis of the e x p e r i m e n t s

responsiveness

43 t I0

_

most

of canine

described

suitable

for testing

lymphocytes

to 100 pl.

in PHA.

of RPMI-1640

of PHA and incubated

5% CO 2 atmosphere.

Sixteen hours

above,

before

found the

the in v i t r o Five pl.

containing

for 4 days

we

of w h o l e

various

at 37°C in a humid,

harvesting

0.5 pCi

of

of 3 H - t h y m i d i n e is added to each well. The reason for s e l e c t i n g 100 pl. c o m b i n a t i o n over the 10 ~i. b l o o d volumes is the greater ease

in o b t a i n i n g

harvesting given

complete

procedure.

as high or higher

control

counts

stimulation

P~A dose Figure

of the red b l o o d the

stimulation

higher,

10 ~i.

w i t h PHA,

thus giving

cells

b l o o d volumes

during

the b a c k g r o u n d

essentially

the

may have or

the same

ratios.

response 4 shows

the o p t i m u m presented

were

lysis

Although

the

the PHA dose r e s p o n s e

conditions

the CPM per

described 5 ul.

of

15 normal

for the test.

adult dogs

The results

blood and s t i m u l a t i o n

ratio.

are

using

5)/1.;

258

ooB

A

I0 4

250

103

150

101

5O

,~ &, & ~

& ;~ ,,~ 0:5 o~,, ;, o.~ o~., &,

~0

25

I0

PHA-P ( / ~ / r n l )

Fig.

2.5

Io

0.5

0.25

O. I

4 PHA dose response in 15 n o r m a l a d u l t d o g s . (A = C P M / 5 ul. blood; B = Stimulation Ratio). Each point represents the mean + standard error of the mean.

Table

III

conditions,

TABLE PHA

5

PHA- P (/~-9/ml)

0

20

the

normal

PHA

dose

puppies

response,

using

between

the

ages

of

16 w e e k

old

puppies a

III

dose

PHA-P

illustrates in

response

in

normal

(ug/ml)

8 to CPM

S.R. b

41

+

25

1260

+

287

27

+

8

10

4430

+ 722

90

+

15

5

7047

+

893

150

+

25

2.5

6844

+

1041

136

+

21

1

2582

+

771

52

+

12

0.5

1118

+

313

22

+

6

a n = 20 b S.R. = Stimulation Ratio c mean + standard error of

the

8c

mean

---

the 8 to

same 16

cultur~

weeks.

259

P W M dose r e s p o n s e Table

IV d e m o n s t r a t e s

the PWM d o s e r e s p o n s e in n o r m a l adult dogs

and p u p p i e s u s i n g the same c u l t u r e o c n d i t i o n s

as d e s c r i b e d

for PHA.

T A B L E IV PWM d o s e r e s p o n s e in n o r m a l adult dogs and

8 to 16 w e e k old p u p p i e s

Adults a PWM

(dilution)

0 1:5 i:I0 1:50 1:100 1:500 1:1000 1:5000 1:10000

Puppies b

CPM 28 5677 5786 5217 4497 3302 2382 786 412

S.R. c

+ 6d + 354 • 904 + 1337 ~ 1368 + 634 ~ 552 ~ 97 + 72

207 212 190 164 118 88 29 13

CPM

--+ 30 ~ 36 + 50 ~ 43 + 29 ~ 22 + 6 + 2

37 +

S.R. 8

---

5700 + 666

138 + 24

5911 + 780

196 + 38

1799 + 780 iii +

44 +

3

3 +

1

17

a

n = n = c S.R. d mean b

8 14 = S t i m u l a t i o n Ratio + s t a n d a r d error of the m e a n

DISCUSSION In a s s e s s i n g

the c e l l - m e d i a t e d

immune c o m p e t e n c e of a p a t i e n t

using the l y m p h o c y t e t r a n s f o r m a t i o n test,

it is e s s e n t i a l to test the

l y m p h o c y t e r e a c t i v i t y not only to o p t i m a l c o n c e n t r a t i o n s mitogens,

but a l s o to s u b o p t i m a l

and Schecter, requires

1976).

relatively

technique described

To a c c o m p l i s h this u s i n q c o n v e n t i o n a l m e t h o d s large v o l u m e s

of blood.

in this p a p e r p e r m i t s

mitogen concentrations ml. of blood.

performed

The m i c r o w h o l e b l o o d

e v a l u a t i o n of up to 60

in t r i p l i c a t e w i t h as little as one

This n o w allows one to e x a m i n e other p a r a m e t e r s of

the immune s y s t e m w i t h o u t t a k i n g copious a m o u n t s animal,

especially young puppies

immune d e f i c i e n c i e s

and small dogs.

of blood

of immune f u n c t i o n tests

It also p e r m i t s

for m o r e

from the

Since m o s t p r i m a r y

o c c u r in the r e l a t i v e l y y o u n g animal,

miniaturization

without

of the

c o n c e n t r a t i o n s as well° ( O p p e n h e i m

the

assumes much importance.

f r e q u e n t m o n i t o r i n g of i n d i v i d u a l a n i m a l s

c o m p r o m i s i n g the animal.

A n o t h e r m a j o r a d v a n t a g e of this t e c h n i q u e in s e t t i n g up of the test. whole blood,

is the r e l a t i v e ease

Since the test is b a s e d u p o n the use of

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

is e l i m i n a t e d .

The s t a n d a r d m e t h o d

separation

for the s e p a r a t i o n of l y m p h o c y t e s

260

from w h o l e

blood

Hypaque-Ficoll technique Since

this

criticism cellular

autologous a volume

fresh

(Felsburg

1976).

in saline.

which

stimulatory

has p l a g u e d

of g r a n u l o c y t e s

as well

lymphocytes

differences

granulocyte

In summary, developed

and

(1973)

We have blood

the w h o l e

cell-mediated

equal

in to

the same

technique

did not alter

In our

blood

(Oppenheim

to m i t o g e n s

studies,

lymphocyte

a new m i c r o

the

reported

found

which

and Schecter,

often

their

surpasses

ability

we have

to

found no

transformation

greatly

immune

in

lympho-

in dogs with

concentrations.

lymphocyte which

technique

of the

have d e m o n s t r a t e d

in the PHA or PWM r e s p o n s e s

and s t a n d a r d i z e d

of the canine

al.

as those of others,

to PHA.

optimally

dog p l a s m a

on the a b i l i t y

stimulation

responses

respond

factors.

and r e m o v i n g

data).

to m i t o g e n i c

Our results,

intrinsic

to PHA of w h o l e b l o o d

our m i c r o w h o l e

unpublished

those of p u r i f i e d

varying

et.

a potential

can be r e c o n s t i t u t e d

normal

or after w a s h i n g

criticism

significant

due to h ~ m o r a l

the cells

or p o o l e d Pellegrino

in the dog using

this

of the serum,

between

the blood

response

to r e s p o n d

ver good

the c u l t u r i n g

responses

After washing

is the e f f e c t

p r o b l e m with

of a u t o l o g o u s

in the s t i m u l a t o r y

et al.,

Another

upon

by w a s h i n g

autologous

on

yield.

is it can not d i f f e r e n t i a t e

p l a s m a volume.

no d i f f e r e n c e

to be true

is b a s e d

or d i m i n i s h e d

plasma.

centrifugation

The p o t e n t i a l

in the p r e s e n c e

can be r e s o l v e d

the s t a r t i n g

cytes

cells

of either

cultured

1968).

technique

defects

gradient

to the r e c o v e r y

of the test

problem

general

(Boyum,

is r e l a t e d

unfractionated

This

is by d e n s i t y

facilitates

competence.

assay has b e e n the e v a l u a t i o n

261

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