A new method of measuring the deformability of the ...

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1975 45: 581-586

A new method of measuring the deformability of the red cell membrane BS Bull and JD Brailsford

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A New

Method

of Measuring

ofthe

Red

Red

cells

be

arrested

means

J. Douglas

which

is successful

a

of surface

THE to those

physical characteristics the formulation of physical attributes. such

deformability,

area,

would

Brailsford

scanning electron microscopy. Preliminary results obtained by this method show elastic strains of nearly 300% and indicate that the stress required to produce these large strains is an order of magnitude greater than that reported by other methods.

network

in duplicating

cell as its marked

tion

of fluid

of

EASUREMENTS OF brane are a prerequisite attempts to duplicate

which

red

in a stream

by

Deformability

Membrane

can of fibrin threads. Those cells which fold over a fibrin thread present to view a flat straplike pertion of membrane in which the strain can be easily observed and accurately measured both optically and by

M

moving

Cell

S. Bull and

By Brian

the

specific

elasticity,

significantly

of the red any membrane A membrane

physical and

memmodel model

characteristics

incompressibility,

narrow

cell

of

and

clarify

the

conserva-

discussions

of

red

cell membrane ultrastructure. Any proposed arrangement of membrane macromolecules would have a lengthy list of physical characteristics to explain, in addition to the many biologic functions which the red blood cell membrane is already known Determining midable branes, practical

to subserve. the physical

technical for drying purposes

characteristics

problems. is known this limits

of

a red

cell

membrane

poses

for-

The measurements must be made on wet memto alter drastically membrane characteristics. For observations to light microscopy of red cells sus-

pended in isotonic diluent. The red cell must then be deformed by a measurable and controllable force which, in turn, requires some method of holding the cell during the deformation. The means chosen to arrest the cell and the method by which the deformation is produced ideally should be atraumatic to red blood cell membranes

and

should

series

can

then

of

forces

optically. since the visible The

Even so, dimensions

light. earliest

permit be

clear

applied

the experiments of the red cell

comprehensive

visual and

observation.

the

A gradually

resultant

will strain the limits are so small relative

estimate

of the

varying

deformation

physical

measured

of light microscopy, to the wavelength

characteristics

of the

of red

blood cell was made by Rand’ using the micropipette technique of Mitchison and Swann.2 In this method, a micropipette is used both to immobilize the cell and to deform the membrane. A known negative pressure is used to aspirate a

From

the

Medicine, Health

Department and

the

Professions,

of Pathology

Department Loma

of

Linda,

Calif

Submitted

June 25, 1974;

accepted

Supported

by a Grant-in-A

idfrom

Address Medicine,

Loma

© 1975

Blood,

for

reprint

requests:

Linda

by Grune

Vol. 45, No. 4 (April),

1975

Laboratory

Medicine,

Technology.

Loma

Loma

Linda

Linda

University

University

School

School

of

of

Allied

92354. October

28, /974.

The Educational Brian

University

& Stratton,

and Medical

School

S.

Bull,

Foundation M.D.,

of Medicine,

Department Loma

Linda,

of A merica, of Calif

Westport,

Pathology

Conn. and

Laboratory

92354.

Inc.

581


582

BULL

hemispheric sure causes lindrical attention

portion ofthe the membrane

red cell into the to move further

tongue with a hemispheric for the measurement

Leblond4

and

shown

that

has

observed. The static pressures deformed membrane is in close across the not known. material

with

membrane (3) The inside

the pipette. observable

wall

features: is axially

which,

membrane.

pipette,

while

(2)

the

ultimately leads of deformation.

the dynamic obtain in the

in the

A

largely

who

“tongue”

maximum

measured and the

avoids

cells

deformation

the

is known

to

and its effect is section of the

occurs

at the

tip

down) which limits by this method to

conditions

interfering

of the material

is established

condition, a small cross

under

rest

of glass,

difference

(necking possible

has

lengths

Only easily symmetrical,

case

pressure

to instability (4) It is not

behavior of the blood circulation.

which

the

is virtually isolated from the however, are: (1) The membrane

pipette

cell

by Evans,5

in producing

itself. This is a nonphysiologic deforming force is applied to

the

This range

A method

the

red

presa cy-

received considerable by La Celle3 and

mathematically

several attractive the deformation

BRAILSFORD

the negative and to form

method has deformability

involved

under observation Disadvantages,

the

The cell

developed are

method has are involved,

contact with

been

deformations

portion material.

interact

serve which

recently

large

end. red

of

pipette. Increasing into the pipette

AND

resembling

effects

of glass

of the ob-

those

has

been

de-

scribed by Hochmuth and Mohandas6and Hochmuth et al.7 Measurements are made on “tethered” cells; that is, cells are attached to a glass slide at one or two points only and deformed by fluid shear. The absence of the constraint imposed by the wall of a pipette tions by this method. certain, uniform Here

folded case

over

of

fibrin

tethered

strands

and

the

whole

cells,

then

be prepared

confirmation cell does not qualitatively

for

of the contact similar

scanning

deformed

extremely is rendered cell

and of

large deformasomewhat un-

the red

by continuous

process

chamber incorporated in a microscope by phase-contrast or by interference quite easily be fixed in the deformed can

to produce the results

however, by the complex shape of the red distribution of stress. we describe a third method, the examination

being the

makes it possible Interpretation of

can

be

carried

markedly cells

arrested

fluid

flow.

out

electron

microscopy,

possible, moreover, to subject cells which they are capable of withstanding.

MATERIALS

to

thus

by As

in an

slide. It can therefore be observed microscopy. Cells deformed in this state while still under the microscope.

optical measurements. any foreign surface, to those encountered

non-

in

optical either way can They

permitting

precise

Under these circumstances, the red and it is subject to stresses which are in the normal circulation. It is the

AND

full

range

of

mechanical

stresses

METHODS

Principle Red occurs into

cells

in a moving

in the a flat

manner

straplike

formation results with the restraining

stream shown

region

of fluid

in Fig. where

from fluid viscous forces produced

the

can

be

arrested

I, each

cell

forms

membrane

material

by two

stationary roughly

actually

fibrin spherical

passes

drag forces which act on the red cell by the fibrin strand.

over membrane

strands. lobes the

When which

strand. and

this blend

The equilibrate

de-


DEFORMABILITY

OF

For

at least

(1) The

flat

two

reasons

straplike

region

measured.

The

area

of membrane

The

cells

Fig.

2. This

fore,

the

brane

MEMBRANE

1 . Stages in the formation over a fibnn strand.

Fig. folded

width

RED CELL

slide

material freely

implies

deforming

where

strap

and

this

the

that, forces

it approaches

must

over

of a double strand

give

the fibrin

rise

the

fibrin

are acting formation

membrane

to almost

strand

is not

pure

tension

be seen

so that can

of

by becoming

amenable

can

of membrane

forces the

arrested

is unusually

the layer

during

at least,

of red cells

of deformation

bends

the ,deforming

the fibrin initially

of an aggregate

type cell

consists

on which

along

release

particular

where here

583

the

easily to

the

in the

straplike

analysis: and

its

cross-sectional

be determined.

constellations

stuck

to clearly

like

strand;

and,

portion

(2)

those

in

there-

of

mem-

strand.

Techniques Red cells

cells in EDTA-anticoagulated

suspended

previously

in autologous

described.8

of 2.5% glutaraldehyde stant flow rate maintained flow was

velocity measured

After

plasma the

whole

blood

were

allowed

“washing

in isotonic saline for 5 mm until

of the blood cell by determining

suspension the velocity

on

obtained

to interact

a line”

from with

aggregates

was substituted complete fixation

fibrin had

healthy strands formed,

human

donors.

The

in a flow

chamber

a fixative

solution

for the red cell suspension had been achieved. Before

and a confixation, the

in the region immediately adjacent to the red cell clump of nearby unattached red blood cells. This was done by

2. Scanning electron microscope of an aggregate of red blood cells arrested by a flbrin strand. x 5000 (reduced). Fig. picture

were


584

BULL

taking

multiple-exposure

attached of flow

red blood can

experiments,

were

found

drying

the

gold

operating

the

The

time

specimen

in the secondary

Triple

little

prepared was

emission

optical

From

flashes of