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1970 36: 153-158

Platelet Cold Agglutinins STANLEY P. WATKINS, JR. and N. RAPHAEL SHULMAN

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From www.bloodjournal.org by on January 7, 2009. For personal use only.

Platelet By

P.

STANLEY

A “cold” platelet which is present

agglutinin in plasma

is

100,000

greater

than

Cold

WATKINS,

Agglutinins

JR.,

is described and serum, in

HE

PURPOSE

molecular

patients.

The

to platelets

agglutination

was

at temperatures

characteristics the

of this report was responsible

that

glutination

of a gamma

automatic

electronic

cably

also

logous

from

a

34#{176}C.Since to room

This

discovered

abnormal

had

to

no

appears

have

of platelet agcounts in five

protein

that

attached

the

physical

and

immunologic

had

no demonstrable

effect

on

five

counts

patients

obtained

were

not

only

by

an

inexpli-

lack of symptoms of thrombocytopenia, but physiologic possibilities. Counts performed

patients

were

clumped,

if blood was not allowed

normal

blood

in microscopic were

platelet

because

anticoagulated

platelets

temperature

an

protein

on

counter1

same

if

or

the

by

34#{176}C and

globulin.

was

the

on

fingerstick

but

34#{176}C. auto-

in vivo. particle

microscopically2

platelets

below and

effect on in vivo platelet function. It is seen in patients with systemic disease and can cause spuriously low platelet counts.

caused

in view of the patients’ from day to day beyond

low

varied

SHULMAN

is to describe a phenomenon for spuriously low platelet

below

function of platelets The clumping agent

RAPHAEL

reacts only at temperatures It agglutinates homologous

weight, appears to be a protein of the immunoglobulin type, is adsorbed by platelets in the absence of bivalent cations, and

T

N.

AND

was

counts an

done

attempt

was

on blood made

diluted directly to cool below that

had

to identify

cooled the

ag-

glutinin. MATERIALS

Anticoagulants citrate

one

were

part

1.4

used

NI

(40

as follows: per

cent)

AND

sodium to

100

METHODS

heparin parts

whole

50 U/mI.

whole

blood;

disodium

blood;

tn-sodium

ethylenediamine

tetraacetate (EDTA) one part 0.27 M (10 per cent) to 15 parts whole blood; sodium oxalate one part 0.075 M (1 per cent) to nine parts whole blood. All blood was drawn in plastic syringes. Platelet agglutination was evaluated by observing the number of platelet clumps in a standard blood cell counting chamber under phase microscopy. The whole blood or mixtures of platelets and protein fractions were diluted 1 to 100 in 1 per cent ammonium oxalate and mixed in a rotary agitator for five minutes prior to observation in the counting chamber. The number of free and clumped platelets could be estimated easily because the clumps usually consisted of two to five platelets and often more than 10. Platelet-rich plasma was obtained by centrifuging anticoagulated whole blood for four minutes at 1000 x g. Platelet buttons for concentrated platelet suspensions were made by centrifuging platelet-rich plasma for 10 minutes at 2000 X g. Resuspensions to the desired concentration were made in ABO-type specific plasma. Serum was obtained by allowrng blood to clot in glass for 24-36 hours at 37#{176}C.Serum was fractionated on C-200 Sephadex columns by standard technique using 0.15 NI NaG! containing 0.004 M EDTA as eluting fluid. The three peaks of material were concentrated From the Clinical Hematology Branch, National Institute Diseases, National Institutes of Health, Bethesda, Md. Submitted January 15, 1970; accepted January 27, 1970. BLOOD,

VOL.

36, No.

2 (Auc.usT),

1970

of

Arthritis

and

Metabolic

153


From www.bloodjournal.org by on January 7, 2009. For personal use only.

154

WATKINS

01 #{149} C

00

C’

C

-

-

-

+

+

0

01

C’1

0 -

O

C

Lt)

C’)

+

o

‘‘

-

0

N

-

Cl

cc 0

t

0

0

o 0

If)

N

N

if)

01

N

.

a

,,

00

N

0

#{176}

a

-

cl

‘‘

z

0001 .0

C’) C’)

EEc

Cl

N 01

Cl

C/D0

N

If)

01 C’)

00 01

0 0

-

0

c

01

N

01

Cl C)

cc

.

If) N

C

E-3

0 ‘

a

..

00

.JC)

f)J

a,

aa

0 -iS

c’i IC)

© 0 S ‘‘

0 0 S 0

Cl

Cl

0 0

0

0

0 0

0

0

0

01

cc

00)

0 oi

-

cc

0

S c#{231} C’1

0 0 S

0 0 S

-

-

1!) C)

S

0 0

C’)

o -

-

-

-

a 0 N C’)

v C

d

C

Ru

il

‘:;

C

C

cc

C)

01

<C’)

-

C)

0

cc

0

cc

C

‘C 0

-

01

C’)

‘‘

If)

0

AND

SHULMAN


From www.bloodjournal.org by on January 7, 2009. For personal use only.

PLATELET

to

one

COLD

half

the

155

AGGLUTININS

original

volume

and

prior to use in the agglutination to 3 mg/mI. in the > 300,000 mol. wt. peak; and 60 mg./ml. and purified proteins the final tions

half

those

Protein an

listed

in

equal

volume

centnifugation against

original

the

<

incubation

400

for

x

g.,

x

105

the

Of

3.0

adsorbed

with

adsorption

anti-human

it

x

in

the

1/32

was

from

10#{176} platelets same

for

manner

as

mixed

with

defined

in

Results

was The

were

was

was

with

incubated

one-third

at room serum.

30

minutes

for 10 minutes. The supernatant sulfate

fluids

Precipitates

that

10

These

minutes. Cryoprotein

used

the by

of

from

The

at

this

were

separated

of

fibrinogen

were

the

with

one-third rabbit

removed

by

for

platelet

for

15

serum

by

M NaCL minutes

at

fluid and

four

was

another

mixtures

centrifuged

mixtures

treated

removed

of 0.15

supernatant

the

with

was

were

globulin After

agglutinin

were

at 25,000

with

antiglobulin

volume

of

anti-human centrifuging agglutinins

X g. serum.

normal

barium

gamma

globulin.

at

25,000

x

as

described

g.

for

above.

by allowing blood to clot at 37#{176}C and then at 4#{176}C.Precipitate that formed in the cold was in a volume of normal serum equal to the volume

the

serum

hours

precipitate

was

degradation

and

of each

excess tested

resuspended

the

of Ferreira

were then

from

48-72

for

which

neutralize

stage

preparations

centnifugation,

presence

techniques

formed

incubated

to

centrifuged

by

adsorbed

serum

in 0.3 ml.

then

5#{176}C,they in

was

adsorption

aliquot

agwhen

neutralized

human

gamma

An 1/8

of the

serum

ml.

containing

above.

specificity

of

for

serum.

precipitate

then

serum

serum

separated

were

at

plasma

completely

suspended

rabbit

minutes

visible

human

was

incubating

serum

removed

30

ml.

at

0.3

approximately

Control

were

the

minutes

titering,

described

to

the

0.6

Platelets

and

normal

at 34#{176}Cand

This

adsorbed

of

as

to determine in

10

In

platelet-rich

and

temperature.

10 minutes

volume

of

concentration used

to

of various concentrathe agglutinin. After

centrifuged

decreased

agglutinin

were

ultrafiltration

titered.

evaluated

The suspensions were heated at 50#{176}Cfor 15 minutes, 2000 x g. (keeping the temperature at 50#{176}C ). One mixed with one-third volume of rabbit anti-human aliquot

of platelets containing

ml.

by

solution

obtained.

plasma was

with

separated

precipitate by

been

mixing

was

concentrated

samples

0.05

final

globulin platelets.

and

had

the

agglutination

g. for

and

supernatant

agglutinating

x

purification

by

precipitate

supernatant

NaG!

temperature

30 minutes

2000

at

further

precipitated

was The

by adding 0.1 ml. 0.3 ml. of plasma

to

was as

peak

protein

and

gamma

eluted

centrifuging

M

platelets at 105/mm.3 2 x 10 platelets/mm.3

with

Rabbit 3

plasma

titered

wt. the

the

in the

platelets/mm.3,

that

0.15

room

for

concentration

sulfate.

Both

performed plasma at

procedure

of protein was adjusted mg/mi. in the < 300,000 to > 100,000 mol. wt. peak. In mixtures of platelets was 105/mm.3 and protein concentra-

25

mol.

which

agglutinin

same

sentence.

300,000

of

from

hour

the

supernatant

glutinin

after

one

and

peak;

water.

volumes

Adsorption studies were tions suspended in EDTA

the

final

ammonium in

serum

wt.

previous

dissolved

of

by

The

in the < 100,000 platelet concentration

saturated

1000

volume

mol.

100,000 of

and

dialyzed

in

>

the

reeluted

mixtures.

obtained,

products

and was

tested

evaluated

for

platelet

by using

agglutinins.

the

immunologic

Murat.3 RESULTS

The

patients

studied, detectable

are

sociated Platelet

with high clumps

Fig. ture

with

listed serum

1. Agglutination in the patientsâ&#x20AC;&#x2122;

fractions

with

platelets

increased

agglutination

curred

low

gammaglobulin that developed was whole

and

levels in the

discernible

blood

suspensions was

above

inexplicably

variable

platelet

counts

in Table 1. Two patients had metastatic protein abnormalities, and three patients

of

34#{176}C.After

in

within

for samples

agglutination

platelets. more kept

than below

occurred

patients

are

of the

patientsâ&#x20AC;&#x2122;

at room serum

The

number

of

several

or in mixtures normal

progressively noted

and cryoproteins. blood of these

whom

minutes

one

hour

no as-

shown

in

temperaor serum

agglutinated

(Table

2).

no

agglutination

at temperatures

below

31#{176}C,but

we

carcinoma but had diseases

Maximum

oc34#{176}C,


From www.bloodjournal.org by on January 7, 2009. For personal use only.

156

WATKINS

AXD

SHULMAN

.,

I,

‘:1

o

.1’.

ii

:1

,, .

L

I

4

Fig. 1.-Platelet phase microscopy ammonium

2.-Rate

Incubation

Number

Time

of agglutinated

Whole indicated

blood times.

agglutinated

blood

at

it could

tain

not accurate

their

blood

diluting

fluid

from

patient

4, Table

of Platelet at Room (22’C)

Agglutination

EDTA

as an

Sera from by Sephadex

103/mm.3)

4, Table platelets

from

37#{176}Cshowed

the

total

no

evidence

be reversed by platelet counts samples

above

used

in cell

effect on the had no effect experiments

at Room

Temperature

(22#{176} C)

Minutes

(x

platelets

5

0

17

15

50

80

120

156

174

to stand at room by subtracting

present

incubation.

of

before clumping

over

the agglutinates patients in Table

34#{176}Cby counts.

0

1, was allowed were calculated

warming on the

the remainder of the counting without agglutination occurring. The types and amounts of had no surfaces glutination

1.

Temperature

from patient Agglutinated

platelets

kept

:

Agglutination. Platelet clumps in cell counting chamber under at room temperature. Whole blood at 1/100 dilution in 1 per cent

oxalate

Table

WilL

warming

After procedure anticoagulants

blood could

the

syringes,

diluted

listed

time

aliquot

of

198 for the of non-

the

same

interval.

to 37#{176}C.In order to ob1 it was necessary to keep

was be

temperature the number

An

this

360

done in

bottles

sample

in counting at room Materials

and

pipettes, temperature

and

Methods

rate or degree of agglutination. Siiconized or plastic on agglutination compared to glass. All subsequent agdescribed were performed in glass test tubes with

anticoagulant. four of the filtration

five patients as described

and from normal controls were in Materials and Methods.

fractionated The aggluti-


From www.bloodjournal.org by on January 7, 2009. For personal use only.

PLATELET

COLD

nating

protein

1, 3 and serum,

not

was

4, and

found

agglutinin

found its

in patient titer.

antihuman

Methods.

After

excess

the

antiglobulin

platelet

Three

wt.

from

of

the

be

agglutinin

75

gammaglobulin

peak

in patients

normal

serum

did

platelets and

best

with

specific

from

this

Materials

)

.

preThe

antisera

be-

and

under

mixture

and

was

platelets

described

from

and Methods

mixed

Materials

was

)

Methods

and

removed there

,

and was

no

remaining.

patients

were

decrease from the the

by

Materials

eluted

as

( see

five

analyzed was

neutralized

Moreover,

agglutinate

4 could This

activity

out

(

resulting

was no detectable was separated

Methods.

identically

be adsorbed sulfate see

precipitate

agglutinating

There protein

prepared

protein could cent ammonium

high

rabbit

with

platelets

mol.

wt. peak in patient 5. As with whole serum fractions occurred only at tem-

platelets.

agglutinating by 50 per of

mol. by the

34#{176}C.Fractions

agglutinate

cause

> 100,000-<300,000

300,000

>

of

below

The cipitated

in the

in the

agglutination

peratures

157

AGGLUTININS

noted in the patientsâ&#x20AC;&#x2122;

cryoprecipitate,

to have

a cryoprotein

platelet sera as

agglutinin described

redissolved

in

their

sera.

titer when in Materials

in normal

serum,

cryoand did

not

platelets.

None

of

the

sera

tamed

detectable in Materials and

or

fractionated

fibrinogen Methods.

material

degradation

that

agglutinated

products,

when

platelets tested

as

con-

described

DIsCusSIoN

A number

platelets

of

substances

in vitro,

of

namely

low

molecular

thrombin,46

weight

are

adenosine

known

adenosine triphosphate, norepinephrine, 5-hydroxytryptamine hydrochloride.6â&#x20AC;&#x2122;102 The agglutinin we are describing weight substance (> 100,000 mol. wt.) as determined filtration. The agglutinin is not dependent on the presence nesium

ions

for

agglutination. described described after

intravenous

et al. was

clotting, divalent sistent proved

The

herein does

of agar,

present

is present require

acid

agglutinin

appears

pH

providing for

activity,

on platelets, immunoglobulin by

to have

has

effect

citrate,

on

factor

Yamazaki

et

rabbit

or adrenalin.

test.3 blood

al.,14

plasma The

same

in acidified described by

oxalate

and

effect

The

cold

is kept reacts

physicochemical type, and in

immunologic

no

no

or EDTA

Kowalaski,16 in 1967, noted that products caused platelets to agcould not be detected in the sera

flocculation

in serum

has

substance appeared The substance

to which

products

sensitive

tin,9

agglutinating

endotoxin

absent in serum. degradation

a highly

not

used

thrombocyte

in plasma

was also of fibrinogen

cations, is adsorbed with proteins of the to be a 7S gammaglobulin cold

anticoagulant

bacterial

degradation

using

described

of

the

agglutinate

triethyl

and epinephrine is a high-molecularby G-200 Sephadex of calcium or mag-

et al.13 and from ADP.8 In 1967, substance present in heparinized

not

and

Fibrinogen

studied,

type

from

that a similar platelet-clumping obtained from normal rabbits.

been added, concentrations

glutinate.

the

differs

injection

noted plasma

Yamazaki

we

and it

by Brinkhous a platelet-clumping

authors15 (pH 5) had low

activity,

Thus,

to

diphosphate,7â&#x20AC;&#x2122;8

agglutinin

at 37#{176}Cduring in

the

absence

properties one instance

of conwas

techniques.

on

platelets

in vivo,

probably


From www.bloodjournal.org by on January 7, 2009. For personal use only.

158

WATKINS

because

it does

have

been

react

best

the ing

not

attach

described at

37#{176}C.17 Extensive

five patients times (Ivy

glutinin

studied

and

none

to

be

agglutinin

spuriously

is

low

and

performed

by

fluctuating

platelet

should

he

was

fluctuating

count

disease

platelet

may

counts,

counter.

If

visual

rather

than

made

on

the

determination

kept

one

bleedag-

to

the

patients.

responsible

especially

particle

blood

five

be

of

The

similar

of the

it

is erroneous,

and

or on venous

that

four

disease.#{176}

in three

in

on

or abnormal

of

present

antibodies

performed

systemic

manifestation

agglutinins

platelet

bleeding, had

importance

electronic

employed,

fingerstick

abnormal

SHULMAN

cold

most

were

patients

which

widely

an

five

clinical

but

procedures

a nonspecific

of

34#{176}C.Platelet

states,

had

All

of cryoprecipitate

The

above

surgical

technique).

appears

occurrence

at temperatures

in thrombocytopenic

AND

for

if counts

suspects

that

a

electronic blood

are

or

low

techniques

obtained

from

a

at 37#{176}C.

REFERENCES 1.

Bull,

B.

Brecher,

S.,

C.:

Counter.

Schneiderman,

Platelet

counts

J.

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with

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Path.

NI.

A.,

the

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and

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G.,

and

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enumeration

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Mason,

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cells

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113-115,

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Adenosine

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1-117,

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ase,

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Brinkhous,

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Platelet Role

Thromb.

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Thrombosis.

Physiology

Vox

E.

1965.

cyte

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clot

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Rodman,

serum

tin.

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clumping

12.

B.:

and

33:161-165,

aggregation.

fibrinolytic

Viscous and

1:133-154,

5.

in

of

F.:

platelets

L.

tn-ethyl

1963.

platelet

Mitchell,

Platelet

demonstrating

9:299-310,

Luscher,

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for

products

Haemat.

blood

Murat,

method

degradation

Brit.

and

B.,

and

11.

and

16:223-226,

Troup,

10:78-93,

An

of

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human

1950. 3.

thrombin

Path.

Hemostasis

Morphology

its

cliphosphate,

Gun. 10.

44:678-688,

1965. 2.

sine

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on

6:641-668,

one

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in

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I.

man.

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1961.

liposarcoma

and

one

with

a


Criogalutininas e contagem de plaquetas