ABSTRACT
A SEARCH FOR BIOLOGICAL EFFECTS OF MAGNETIC FIELDS

By Adolph Eric Smith

The potential importance of influencing biological pro—
cesses by magnetic fields, in conjunction with the long and
controversial history of biomagnetism led us to undertake a
careful study of some well defined and sensitive phenomenon
of biological significance. We have selected the immune re—
action, specifically the antigen-antibody reaction as evidenced
by agglutination of human erythrocytes. In this thesis we
first review the physics of magnetic fields and suggest
possible ways in which the small energy of magnetic inter-
actions——as contrasted with thermal effects-~might have de-
tectable effects in biological systems. Laboratory-scale
magnetic fields would appear to be capable of only very
slight action on individual atoms or molecules, but should
be able to produce significant effects on groups of associ—
ated molecules such as occur in liquidfcrystaline or other
mesomorphic states. Next we review the background of immuno—
logy relevant to the present problem with emphasis on the Rh-
blood—group system.

To assess the effects of magnetic fields on agglutin-
ation, quantitative scoring methods are necessary. Three
methods were considered in the present work: visual scoring,
in which the agglutinated material is examined visually for

clumping under the microscope; sedimentation rate, in which

Adolph Eric Smith

the increase in r"te of sedimentation is obscrved when
agglutination takes place; particle-size distribution, in
which the change in size distribution of particles upon
agglutination is observed.

With visual scoring, the magnetic field was observed
to enhance the agglutination in the Rh system, but not in
other groups studied. In the sedimentation studies, only
preliminary experiments were carried out to test the
feasibility of the principle; no experimental results are
yet available. With particle-size distribution, which were
made with an electronic counter, the method had to be
developed. A study of the counter operation and of erythro-
cyte distributions was first made. In a suSpension of un—
agglutinated erythrocytes, the volume distribution of normal
erythrocytes in saline was found to follow a lognormal
distribution. Upon agglutination the distribution changed
significantly. Several statistics were investigated in
order to characterize the change in distribution upon
agglutination; one of the more useful ones was the ratio of
doublet particles to the singlets. This statistic was
studied as a function of incubation time, antiserum concen-
tration, and temperature. This statistic failed to show the
difference between test samples incubated in the magnetic
field and control samples incubated outside the magnetic
field. We present a tentative explanation of the discordance
between the results for the visual method and for the counter

method. The explanation is based on the difference in

Adolph Eric Smith

treatment of the samples in the two methods.

Finally, the significance of magnetic effects for
possible elucidation of the agglutination mechanism is dis—
cussed and extension of the magnetic-field studies for this

purpose is urged.

A SEARCH FOR BIOLOGICAL EFFECTS OF MAGNETIC FIELDS

By

ADOLPH ERIC SMITH

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physics and Astronomy

1963

l I

:3 (.2 v; 5/

///ofl&§

ACKNOWLEDGMENTS

I would like to express my deep appreciation to
Professor D. J. Montgomery for his constant encouragement
during my years as a graduate student in the Physics
Department. His patience and understanding have helped me.

Professor Emanuel Hackel has made valuable sugges—
tions in the writing of this thesis and in the course of
the experimental work.

I would like to express my thanks also to: Messrs.
Thomas Tucker, Jan Schoneman, Kiwun Kim, and Miss Margaret
Case for their help in the experimental work; Misses

Charlane Temple and Diana Lee for their help in calcula-

tions; Mr. Harold Pawlowski for construction of the magnetic
field measuring equipment and field mapping; and Mr. Charles

Kingston and the shop for construction of much of the equip-

ment used in the experiment.

The financial support for this came initially from

the All University Research Fund and later from the National

Institutes of Health.

ii

TABLE OF CONTENTS
Chapter
I. INTRODUCTION

Background of Biomagnetism

Energy of Interaction of the Magnetic
Field . .

Possible Biological Effects of the
Magnetic Field . . . . .

Background of Blood Grouping

Statement of the Problem . .

II. EVALUATION OF THE EFFECT OF MAGNETIC FIELDS
ON AGGLUTINATION: VISUAL SCORING

Materials and Procedures
Magnetic Fields

Visual Evaluation
Results

111. EVALUATION OF AGGLUTINATION: SEDIMENTATION
TECHNIQUES . . . . . . . . . . . . . .

IV. EVALUATION OF THE EFFECT OF MAGNETIC FIELDS
ON AGGLUTINATION: PARTICLE SIZING

Introduction

Description of the Coulter Counter and
Particle— Size Distribution Plotter

Volume Distribution of Erythrocytes

Choice of a Statistic to Characterize
Agglutination .

Dependence of D/S on Magnetic Field
Strength B . . . . . . . . . .

V. DISCUSSION
Visual Method

Counter Method
Reconciliation of Results

Possible Mechanism for Effect of Magnetic.

Field on Agglutination . . . . . . . .
VI. REFERENCES

APPENDIX

iii

Page

ll
14
22
4O
42
42
46
49
58

6O
60

61
66

76
86
92
92
92
92
94
97

101

LIST OF TABLES

Table Page
I l DiSputed Magnetic Effects . . . . . . . . . . 3
2 Effect of Antiserum Concentration on Anti-D
Reaction with D-Positive Cells . . . . . . . . 54
3 Enhancement of Agglutination by Magnetic
Fields . . . . . . . . . . . . . . . . . . . . 55

4 Effect of Magnetic Field on Anti-D
Reaction with D+ Cells . . . . . . . . . . . . 56

5 Window Counts for Sample of KK Blood . . . . . 68

6 Chi-Square Table for Runs 3-11 of Table
5 . . . . . . . . . . . . . . . . . . . . . . 69

7 Reliability of (6/5) . . . . . . . . . . . . . 77

iv

Figure

10

ll

l2

l3

14

LIST OF FIGURES

Illustration of the force exerted by an
inhomogeneous magnetic field on current
elements . . . . . . . . . . . . . .

Linear arrangement of molecules in the
mesomorphic state

The essential composition of blood

The permanent-magnet fixture in the
water bath . . . . . . . . . . . .

The Model R—3 electromagnetic with the
temperature fixture in the field

The Model L-128 electromagnet with
test-tube holder in position

Map of the magnetic field at the
position of test tubes for agglutin-
ation studies

Plot of magnetic field Quantities as

a function of distance 2 from apex

of iron wedge placed in gap to produce
inhomogeneous field . . . . . . . .

The test-tube holder used to position
the test tubes in the field of the
large electromagnet . . . . . . . .

Schematic drawing of the Coulter Counter.

Volume distribution of agglutinated
erythrocytes as obtained with Coulter-
counter plotter . . . . . . . . . .
Volume distribution of KK erythrocytes

Logarithmic probability plot of volume
distribution shown in Figure 12

Plot is similar to that of Figure 13

V

Page

17
26

44

45

47

48

50

51
52

63

71

72

74

Figure Page

15 Semilogarithmic plot of ratio of spike
height upon agglutination at various con-
centrations as shown, to spike height
for normal unagglutinated cells, as a
function of particle volume (proportional
to window number) . . . . . . . . . . . . . . . 75

16 Agglutination, as measured by D/S, as a
function of concentration for D-positive
cells incubated with anti-D serum for 45
minutes at 37.50C . . . . . . . . . . . . . . . 78

17 Agglutination, as measured by D/S, as a
function of incubation time for D-positive
cells incubated with anti—D serum at 37.50
at two concentrations . . . . . . . . . . . . . 8O

18 Agglutination, as measured by D/S, as a
function of concentration for D-positive
cells incubated with anti-D for 45 minutes,
at two incubation temperatures . . . . . . . . 81

19 Counts in various windows representing
singlets, doublets, triplets, and quadrup-
lets for various concentrations of anti-D
serum . . . . . . . . . . . . . . . . . . . . . 83

20 Agglutination, as measured by Q/S, as a
function of concentration for D-positive
cells incubated with anti—D for 45 minutes
at 37.500. . . . . . . . . . . . . . . . . . . 84

21 Agglutination, as measured by Q/S, as a
function of incubation time for various
concentrations of anti-D . . . . . . . . . . . 85

22 Correlation between visual data (as scored
on the Race scale) and the counter data (as
measured by D/S), for the same samples . . . . 87

23a Semilogarithmic plot of the ratio of spike
height for test samples incubated in mag-
23b netic field to Spike height for control
samples incubated outside magnetic field,
as a function of particle volume (window
number) . . . . . . . . . . . . . . . . . . . . 89

24 Agglutination, as measured by D/S, as a
function of concentration for D-positive
cells incubated with anti-D for 45 minutes
at 37.50C, with magnetic field on and with
magnetic field off . . . . . . . . . . . . . . 91

vi

I. INTRODUCTION

Background of Biomagnetism

That gravitational fields, electric fields, and concen-
tration gradients affect biological processes is unquestioned.
But the action of magnetic fields, a fundamental force in the
universe, is not even considered in many physiology texts.
Indeed, the existence of magnetic effects has generally been
denied by most scientists for nearly a century and a half.

Recently, however, there have been reports that magnetic
fields have profound physiological effects. Supporters of
the existence claim that since a uniform field can rotate
magnetic dipoles, and an inhomogeneous field can pull them,
it is natural to expect effects. The opposition claims that
the thermal energies will swamp any effects due to the mag-
netic field. At present, there is no accepted answer to
the question of whether biological effects of magnetism

exist.

Historical

 

Standard works on physiology contain either no mention
of the effect of magnetic fields on biological processes, or
the statement that magnetism cannot be considered a stimulus
for biological processes. Bonner (l), in his essay on

morphogenesis, states: ”There is every evidence that no

living precess is much affected by reasonable magnetic
forces." Heilbrunn's text (2) on physiology is cited in
support of this view; it states:

The above list [of stimuli] includes no mention
of magnetic forces. According to Verworm (3) (1899)
these have no effect on living systems and, there-
fore, cannot be considered stimuli. In support of
this opinion he cites the extensive experiments of
Peterson and Kennelly (4), done in the Edison Labora-
tory in 1892. These investigators used very strong
electromagnets, but were unable to detect any
noticeable effect on living material.

In the book of Verworm, which includes a similar list of
stimuli, is the observation:

. . in this enumeration magnetism is wanting. But
it is now known with certainty that magnetism exer—
cises no effect whatever upon living substance, and
cannot be properly termed as stimulus. . . . Careful
experiments upon the influence of magnets upon the
living organisms have always yielded negative re—
sults. The recent extended researches with very
strong electromagnets by Peterson and Kennelly in

America demonstrate the utter ineffectiveness of
magnetism upon living matter.

Yet, despite these sweeping statements, there are cases
where a magnetic field has been reported to have produced
observable effects on biological processes. Table I lists
some of the divergent findings on the subject. Here is a
curious situation wherein different investigators have ar-
rived at different conclusions about the same subject.
Regrettably, the field strength, the field uniformity, the
duration of eXposure, and the Species tested were not

exactly the same in the cases of diSpute.

TABLE 1

DISPUTED MAGNETIC EFFECTS

 

 

fl
Effect Affirmative Negative
Yeast and bacteria Kimball (5) 1. Leusden (7)
growth Barnothy (6) 2. Jennison (8)
Tissue-culture l. Huzella ( 9) Payne-Scott (13)
growth 2. Lengyel (10)
3. Lenzi (11)
4. Mulay (12)
Mouse growth Barnothy (l4) Eislein (15)

 

There are in the literature, however, accounts of
several affirmative results that are uncontested to date:
(1) Magnetic phosphenes: Thompson (16), Magnusson (l7),
Barlow (l8); (2) Magnetic compass effects on snails: Brown
(19); (3) Magnetotropism of plants, Audus (20); and (4) Grow-
ing speed of plants: Ssawasotin (21).
But also in the literature are some undisputed accounts
of negative results with magnetic fields. These are:
l. The perception of steady magnetic fields by
humans: Peterson and Kennelly (4), Beischer
(22).

2. Attempt to influence the asymmetric chemical
synthesis characteristic of living systems:
Pasteur (23).

Detailed investigation of such phenomena shows us why
it is difficult to obtain conclusive results. First of all,
the effects appear to be slight, and hence the experiments
must be highly refined, or a vast number of replications must
be made. Secondly, theoretical considerations based on

simple physical phenomena fail to give much indication as to

how magnetic fields might produce measurable effects on

living processes.

Hence we shall expect that experiments of a high order
of refinement or very high degree of replication, will be
necessary to demonstrate any effect; and we shall expect
that the theoretical explanations for any effect will depend
upon complex interactions. These ideas will be developed in

the body of our thesis.

Physics of the Electromagnetic Field (24)

When a particle interactswith other particles within
a system, it is often found that parameters other than mass
must be ascribed to each particle. For a certain class of

phenomena one of these parameters is the electric charge,

 

which may be of two different kinds, labeled positive and
negative. In describing the behavior of the particles, it

is convenient to introduce certain mathematical auxiliaries
called electromagnetic field vectors. The part of the inter-

action depending upon the relative positions of the inter-

 

acting changes is described in terms of the electric field,
characterized by the electric field strength E and the
electric diSplacement D. The electric field is set up by the

charge according to the relation;

d2 _'-'- ,0 dV _1_'_/r3
where r is the position vector from the point under consider~
ation to the charge element consisting of the volume element

dV with charge densitylp. The part of the interaction of

the velocity Q'of the particles is described in terms of the

u"
v

magnetic field, characterized by the magnetic induction B and
the magnetic field strength E, The magnetic field is set up

by the motion of the charge according to the relation:

dl/yxr.
P c 73/
where r is the position vector from the point under consider-

d5:

ation to the current element consisting of the current dens-
ity element flyobvand c is the velocity of light. Here we
have assumed shat the fields are changing sufficiently slowly
in time that diSplacement current negligible is compared with
convection current. These two equations, essentially Coulomb's
Law in the electric case, and the Biot-Savart Law in the
magnetic case, form the basis of the calculation for the set-
ting up of the field by the sources.

To see how the fields act on charged particles, we need
the Lorentz equation for the force dfi upon a test particle
of charge dq, a particle whose charge is sosmall that it
does not disturb the previously existing arrangement of charge
and current:

as: = quE + <_v/c> x g]

As the equations now stand, no connection exists be-
tween the pair of vectors 2 and 3 set up by the sources, and
the pair of vectors E and B which exert influence on the
particles. The connection is made through postulating the
following two relationships:

12. = 6 £5; E = 2/11

Here, €n the dielectric constant, and .u, the magnetic

permeability, are equal to unity in free space in the Gaussian

system of units, and take on values different from unity
within material media. In many of the cases of interest‘é
and/q may be taken simply as scalars and, moreover, as con-
stants so far as dependence on D or H is concerned.

We need not labor the matter of the force on charge
elements in the electric case. The direction of force is
quite simply given by the direction of the line between two
charge elements. In the magnetic case, on the other hand,
the forces which depend upon the velocity of the charges--
that is, on the currents or on the magnetic field set up by
these currents, if one prefers-~are more complex. To see
in detail how these forces arise, suppose we first consider
what happens when a rectangular loop carrying a current i

is placed in the homogeneous magnetic field B. Each side of

 

the 100p experiences a certain force, proportional to the
current i, the length of the side, and the magnetic field
strength B; however, this force is exactly balanced by an
equal and opposite one on the part of the loop diametrically
Opposite, so that the net £2553 is zero. There is, of
course, a non-vanishing torque on the loop, given by the pro-
duct of the force and the lever arm.

The more interesting case is that of an inhomogeneous

 

field, wherein the two forces on the opposite sides of the
loop do not exactly balance. To show the main features of
the action, we take the special case where the plane of the
loop is parallel to the xy-plane, as shown in Figure 1. We

see that the force on side 1 is upward, and is given by the

 

 

2 F.
Y
m
i 7'
X -- (4) (3 AX
' <2) .
I
F!

Similarly

AF,“ = --[aBy lay] mz
-[an/ax]-[aBy/ay] = [582/ a2]

so Fz = ”[682. la 2]

In general f =(r_T)-V)§.

Figure 1 - Illustration of the force exerted by an inhomogeneous magnetic
- field on current elements.

product of the magnetic induction in the x direction at side
1, multiplied by the current i times the length of the side
.Ay. Correspondingly, the force on side 2 is proportional
to the induction at side 2; that is, BX2 multiplied by 1
times 41y, but directed downward rather than upward. Now

to first-order terms, the induction at side 2 is equal to the
induction at side 1 Bxl , plus the rate of change of BX
with respect to x, times 11x. Therefore, the net force on
the sides 2 and l is equal to the difference between these
two quantities, that is, —(QBXAéx) i.4y .4x.

The quantity i times the area of the loop, Ay 4);, occurs
frequently in calculations and is given a separate name, the

magnetic moment (this quantity is actually a vector 2, and

 

here we have merely the 2 component, perpendicular to the
area Ay.4x). From these considerations we see that the net

force from the other two sides is equal to C-aBy/Qy) mz.

But now by use of the Maxwell system, ‘7' B = O , we have:
_ an _ aBy = 3B2.
9X ay 62'
hence finally: P2 = mz (aBzfléz). The full analysis

for the general case leads to the following relationship:
§=(_rg'v)l_3_.

-Because the concept of a current loop is somewhat
difficult to visualize, one frequently replaces it by a
fictitious source consisting of a pair of magnetic poles
(which, of course, cannot exist separately in view of the
basic relationship that v - E = 0 ). Nevertheless the ex-

pansion of the actual field set up by a finite current loop

leads to an expression whose leading term has the same mathe—
matical form as that of a field set up by a pair of monopoles
of equal and opposite "magnetic charge” separated by a short
distance. It becomes convenient, therefore, to talk about
the fields of a magnetic dipole, i.e., the idealization of a
pair of equal and opposite poles separated by a certain
distance. The limiting form of the dipole is determined when
the distance between the charges goes to zero, and the charges
become infinite; their product then attains the magnetic
moment as its limiting value. In mathematical treatments the
magnetic dipole is handled in the same way as the electric
dipole. The vast difference in the consequences lies, of
course, in the fact that electric monopoles exist, and at
large distances become the dominant term, the dipole and
higher-order fields becoming negligible. But with magnetic
fields there is never a monopole field, and hence the mag-
netic dipole always becomes the dominant factor. It is for
this reason that much of the analysis is carried on in terms
of the magnetic dipoles.

The form of the field set up by a magnetic dipole of
moment fl oriented in the direction of a positive z-axis is
of the following form:

B = -(2m/r3) cosG Br + (m/r3) sinQ Be
Here r represents the distance from the origin and G the
angle between the point of observation and the axis of the
dipole (zenith angle). fir is the unit vector in the

direction of increasing r, with 9 and fl held constant; and

10

B9 is a unit vector in the direction of increasing 9, with r
and fl held constant. Replacing the magnetic poles by equal

and opposite electric charges giVes a resulting field which
is of the same form as the magnetic field except for changes

in the notation.

Electromggnetic Fields in Ponderable Bodies

When an electric field is applied to a body, there is
a change in the resulting field from that in a vacuum. Since
details of this change are treated in standard texts, we
need not review the results here. When the magnetic field
is applied to a body there is a similar change in the re-
sulting field. We need to discuss this response in more de-
tail. On the atomic level the change in fields results from
the disturbance of the atomic orbitals and the electronic
Spins. The change in the magnetic field is usually described

by introducing the magnetization M_related to B by the

 

following expression:
1.3. = E + 4r 1.1 = w.-
The origin of this relationship is explained in standard
texts. The magnetic Susceptibility X.is defined by:
'X= M/B; whence X = l(}1-l)/477‘

The atomic basis for the magnetic polarizability is the
magnetic moments produced by the motion of the electrons.
The magnetic moment of the electron-orbital motion M2 is

simply related to the angular momentum L by:

M2 = gMB L where

20

M

B Bohr magneton = 0.927 x 10-

erg/gauss

11

There is also a magnetic moment in the atoms due to the
electron spin. Langevin found with classical physics that,
for weak fields:

x: MNZ/kT ------------------------------------ (1)
where N = number of atoms/cm3. For stronger fields the ex-
pression becomes 7C: NM3L (x)/kT, where x S MN/kT and L (x)
E coth x - l/x.

These expressions give susceptibilities that are always
positive. To account for diamagnetism, Langevin considered
the induced magnetic moments of the atoms. Each of the
electrons moving about the nucleus is similar to a current
loop, and by Lenz's law, the magnetic fields induced in this
loop will have a direction which opposes the inducing mag-
netic field. Langevin's formula for diamagnetism is:

2! = -(Ne2/6mc2) E;<ri>2 ---§ --------------------- (2)
where <ri>2 is the average square of distance of the i-th
electron from the nucleus. Diamagnetic susceptibilities
usually range from 10—6 to 10-5. If the molecule has a
permanent magnetic moment, the expressions (1) and (2) have

to be added.

Energy of Interaction of the Magnetic Field
For charges whose velocity is low compared with that
of light--as is the case for all phenomena of chemical and
biological interest--the effect of the magnetic field is
much smaller than the effect of the electric field, at

least in non—neutral systems. To get some idea of the order

12

of magnitude of magnetic effects, let us consider the elec—
trons in an atom.
At a distance of approximately two gngstrom units

(R) from a proton or an electron, the electric field eZ/r
amounts to about 106 volts/cm. The magnetic field, iZnVrB,
at this distance from an atom with a magnetic moment of one
Bohr magnetron, 2 MB/r3, is about 2500 oersteds. The pro-
duct NWOEPéPat 2X from such a dipole is the following:

F”: M (9;?) % Maggi) - 4 (22:07)): ’- o'qx’o-Fdane.

A typical electric dipole moment is about 10'18 esu.

.Iix 2x 4.6')‘ [Odo
(ax/0")?
Now we see that it is possible to obtain microsc0pic

I:

 

35 ~ —
FE -.- [0'37 :10 2:- /0 “cw/gag,
magnetic fields in the laboratory (~104 gauss) as much as ten
times as great as the magnetic fields within the atom

(~103 gauss). On the other hand, the gradients obtainable

 

in the laboratory are not likely to be much larger than

4 gauss/10_1 cm. = 105 gauss/cm. This gradient is smaller

than microscopic ones by a factor of 107. Hence, we see that

10

the forces obtainable macroscopically are negligible compared
with those obtainable microscopically; on the other hand, the
torques obtainable macroscopically are quite comparable with
or even larger than those obtainable microscopically. More-

4 times

over, the microscopic magnetic forces are about 10-
the microscopic electric forces.
To see quantitatively how the externally-applied mag-

netic fields influence the distribution of the atomic moments,

we note that when the field is switched on,the atomic moments

l3

precess about the direction of the applied field, in such
fashion that the projection of their moments on an axis
parallel to the applied field takes on the value +l/2 or
~1/2. In the former case the energy of interaction is de-
creased, in the latter case increased, by MBH. At equili-
brium, the lower—energy state will therefore be slightly
more pOpulated than the higher state, by the ratio exp
MBH/kT to exp -MBH/k13 or 1 + 2MBH/kT, approximately, since
2MBH/kT is small, amounting to about 2/500 at room temper-
ature for a Bohr magneton in a 104 gauss field. We see
then at equilibrium for every 250 moments pointing in the
direction of the field, there will be 249 pointing against
it. In the absence of cooperative effects, it seems un-
likely that this slight alignment-~which, for a given set
of atoms, is continually being disturbed by thermal agitation

--could result in any pronounced biochemical effect.

Cooperative and Statistical Phenomena

Although the energies associated with the external
magnetic field are small compared with thermal energies, and
the field strength is small in comparison with the mascro-
scopic fields, there are still important responses to the
application of the external fields. Ferromagnetism and
ferroelectricity are two striking examples. Here an external
field brings about a slight alignment, but the result of
this new reorientation is to produce alignment of adjacent
molecular groups. These groups then cooperate to produce

a resulting field which may become very strong and create

14

an overall effect which produces stability in the presence of
disrupting forces. It is unlikely that either ferro-
electricity or magnetism is of any interest in biological
systems, although there are certainly some special systems
that are clearly ferromagnetic, such as radulae of Chitons
(Phylum Mollusca) (25). Cooperative phenomena are ex-
hibited by certain large unsymmetrical molecules which group
together to present associated effects which may become
important on the mascroscopic level. These phenomena occur

in the so—called mesomorphic or liquid-crystal state.

 

 

Possible Biological Effects of the Magnetic Field

 

Effect of Orientation

 

Even though a single atom or group of atoms in a typi-
cal molecule could be oriented by the external magnetic
field--and we have seen how small is the possibility for
this orientation-—it is not likely to affect the macrosc0pic
behavior in the absence of a mechanism for coupling this
orientation with the rest of the molecule. On the other
hand, in certain crystals that do occur in the mesomorphic
state, the whole molecule can be oriented by a magnetic
field and can transmit this effect to neighboring molecules.
Since some -. protoplasmic material is known to exhibit
certain properties of liquid crystals, we examine in detail
the mesomorphic state to determine if its occurrence is
sufficiently frequent to permit influencing biological pro-

cesses by magnetic fields.

15

The mesomorphic state (liquid crystals) (26-28)--For
nearly a hundred years it has been known that a few sub-
stances in a restricted temperature region have both liquid
prOperties (such as fluidity) and crystal solid properties
(such as birefringence). This state of matter is known under
various names--"mesomorphic state," ”liquid-crystal state,”
or the "paracryStalline state." Nowadays it is well known
that the bonds between atoms in different molecules may be
of different kinds and may have different orders of binding
energies. Consequently, there are states of matter that are
not described simply by the earlier threefold classification
of solids, liquids, and gases. In general, we may well ex—
pect that if the chemical attractions in different directions
are of varying nature, one :might be able to destroy the
order in one direction, for example, by heat or by a polar
solvent, while retaining the order in different directions.
Indeed it has been found that perhaps 3,000 substances ex-
hibit the liquid—crystal state.

Most of the compounds in the mesomorphic state are
classified as either smectic (like soap) or nematic (like a
string). But a third state of Special interest also exists

in certain biological systems, the cholesteric (like

 

cholesterol, the prototype of this class). In the smectic
state the molecules are arranged with their long axes approxi—
mately normal to the layers in which they are stratified.

In the nematic state the molecules are arranged with somewhat

less order, the only restriction being that the molecules are

16

lined up nearly parallel. In the nematic state the molecules
can be lined up even by weak magnetic fields, whereas in the
smectic state it is difficult to align molecules under the in-
fluence of any except the relatively strong magneticr fields.
In view of the wideSpread occurrence of cholesterol and its
derivatives in biology, this form has special significance.
The cholesterol derivatives as well give cholesterol-type
liquid crystals. The cholesterol liquid crystals have the
property of being uniaxial and having negative birefringence.
These properties are believed to be explainable in terms of
a screw structure for this state. There is no sharp trans-
ition from the cholesterol form to the nematic form, where—
as there is a Sharp transition to the smectic form in the
cases where the material exists both in the smectic form and
nematic form. The optical activity of the cholesterol form
is high; there may be as many as 200 revolutions in one mm.
Figure 2 is a schematic representation of the three forms
of the liquid-crystal state.

Some polypeptides give mesomorphic structures when
they are precipitated from solution,and in these there is a
layered structure which is sometimes twisted into spirals
in spherulites. The polypeptides are of great importance in
biology because the protein structure consists of poly-
peptide chains. In the formation of antibody molecules, the
configuration of the polypeptides is of the greatest im-
portance and it is possible that this may have something to

do with the ideas of the cholesterol-state.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a} nematic state b) smectic state

Figure 2 - Linear arrangement of molecules in the mesomorphic

mm

c)Hellcal array of the molecules in a cholesterol-like
liquid crystal. XYZ is the rectangular coordinate

System; g and 77 are the axes of the molecules.

18

To describe logically the transition between the
crystal and the liquid state we may give the following
description: (1) True (three-dimensional crystals). The
atoms are all fixed at their lattice points and only thermal
vibrations about the lattice points are performed. (2)

Molecular crystals with rotation of the subunits. The lat-

 

tice units are fixed at the lattice points, but rotation
about one or more axes is allowed for the unit. (3) Smectic

structure. The lattice units can move in two directions and

 

rotation about one axis is permitted. (4) Nematic structure.

 

The centers of mass of the molecules are mobile in all
three directions and rotation about one axis is permitted.

(5) True liquid. The centensof mass of the units are

 

mobile in three directions and rotations about three axes
perpendicular to one another are possible.

All the compounds which exist in the mesomorphic state
have been found to be elongated molecules of fairly large
size. In the crystalline state they are rigidly aligned
and as the temperature is raised, a solvent is introduced--
the weaker bonds are at first loosened. The molecules then
have some freedom in certain directions as the thermal
energy or other disrupting forces become great enough to
partially destroy the aligning forces, but not so great as
to destroy the partial ordering forces.

The nematic structure: In the cases where any pro-
gress has been made in understanding the mesomorphic state,

it appears that mobile electrons within the conjugated

19

double—bond systems react against applied magnetic fields

so as to produce diamagnetism. The molecule then sets it—
self up so as to minimize the energy of interaction with the
field. In the nematic state the molecules set themselves
perpendicular to the lines of magnetic force. The diamag-
netic movements for the whole structure and the group of
atoms (typically a hundred thousand) have a resultant inter-

5 times that of a single molecule.

action energy equal to 10
This energy is more than enough to overcome the disruptive
thermal energies. Hence, it is possible for relatively weak
magnetic fields to line up the swarms against thermal
agitation. Because of Space limitations it is not possible
to discuss here the various attempts to produce theories

of the liquid state, which are discussed in several review
articles or books.

The biological importance of the mesomorphic state is
that matter exists in the mesomorphic state in various bio-
logical systems. Recently the deoxyribonucleic acid (DNA)
in living sperm was found to exhibit certain characteristics
of the mesomorphic state (29). The widespread occurrence
.‘~.——-..1",,H.-Mi--i-.in, .--.-----u- . w

of optical birefringence in protoplasm (2) suggests the

occurrence of mesomorphic phases in this material.

Effect of Magnetic Field on Chemical Reactions
Since the magnetic—interaction energies are so Small
compared with thermal energies, it is not expected that

chemical reactions can be influenced by macrosc0pic magnetic

20

fields. Indeed, many experimenters have sought this effect,
but none have shown conclusive evidence (Selwood (30)).
Certain workers (Bahatnagar, g£_3l.(3l)) report positive
effects in the reaction of iron with chlorine. The effects
are very small and, according to Parker and Ames (32), are
made even smaller by stirring the solution during the re-
action. To get an idea for the mechanism, these workers
calculated the difference in concentration for paramagnetic
substances. They took the Boltzmann relationship for
particles in a potential field as (33):

n = nO exp (-WH/kT)
where n is the concentration in the given field,

WH = magnetic interaction energy

no is the concentration in the absence of the field,

N is Avogadro's number.
If we consider molecular oxygeq'whose molar susceptibility 2K
is about 3400 x 10-8 in a field where H = 10 4 gauss

wH=_1_2£flE=_1_X3400

2 N 2 6 x

-6
101% x (101'4)2 2: 3 x 10‘19

 

n = no exp (—WH/kT) 3’ 1.0002 no.
This calculation shows that the magnetic field has a
negligible effect in changing the concentration levels in
a chemical reaction even where so highly paramagnetic a
substance as molecular oxygen is used.

On the other hand, it is to be expected, and indeed

it is observed, that chemical reactions can be affected by

microscopic magnetic fields. The ortho-para hydrogen

21

conversion is known to be catalyzed by paramagnetic sub-
stances. The catalysis of the cis-trans reactions by para-
magnetic substances is attributed by Harman and Eyring (34)
to the differing action of the inhomogeneous field on the
magnetic dipoles which arises from the spin of the two
electrons in double bonds. It is thought likely thatin the
catalysis of certain reaction§,. paramagnetic ions have
their action through the relatively strong magnetic fields

around the highly paramagnetic ions.

Consequences of the Lorentz Force

 

Membrane tran§port.--Consider the effect of a magnetic

 

field on the transport of ions across a membrane. The force
on the ion is given by (y/c) x B, where B is the magnetic
induction parallel to the membrane. The potential difference
across a frog skin, for example, is about 100 mv, and the
thickness of the skin is about 100 microns. The electric
field is then approximately:

E = 0.1/0.01 = 10 volts/cm.

A typical ionic mobility in water is that of sodium:

p 0.135 cm/sec/esu volt (35)

4.4 x 10‘2 cm/sec/volt/cm
A typical drift velocity is then:
v = p.E = .44 cm/sec.
For a field of 10,000 gauss the radius of curvature for a

sodium ion is then about:

R =IEE%‘: 10-7 cm = 10 X
e

22

No effect was found by Knoll (36),who used tagged sodium in
a homogeneous magnetic field of 10,000 gauss. The result
may be due to the sodium-ion mean-free—path being much less
than 10 X.
Background of Blood Grouping (37, 38)

The concept of blood transfusion goes back to ancient
times, but no transfusions were actually carried out until
the middle 17th century. Death often resulted from a trans-
fusion; consequently, the practice was abandoned for nearly
a century and a half. In the 19th century direct transfusions
of blood between humans were attempted. There were many
disasters and the progress toward a satisfactory technique
was slow. In a series of classic papers at the turn of the
century, Landsteiner announced the discovery of the existence
of blood groups, and laid the foundation for safe blood—
transfusion technique.

By 1875 it was known that if red blood cells of an
animal were mixed with serum from an animal of another species,
or sometimes with serum from even the same Species, a clumping

(technically, agglutination) occurred. In 1900 Landsteiner

 

(39) found that the cells of some humans were agglutinated by
the serum of other humans. He took blood from humans, sepa-
rated the serum, and prepared saline suSpensions of the cells.
The cell suspensions were mixed with various sera and some-
times the cells agglutinated. On the basis of his results,
.Landsteiner was able to classify most human blood into three

groups. Within each group the red blood cells of any

23

individual were not agglutinated by the serum of any other
person in that group. In 1902 two of Landsteiner‘s students
discovered a fourth and relatively rare group.

To explain his results, Landsteiner postulated the
presence or absence on the blood cells of two substances
(antigens), now known as A and B. The classification of
people into four groups was based on the following: (1) A
only, (2) B only, (3) both A and B, and (4) neither A nor
B. The groups are known reSpectively as A, B, AB, and O.
In a given blood sample, the serum did not contain the sub-
stances (antibodies) capable of reacting with the substance
on the cells (the antigens). In the following the recipro-

cal relationships are shown.

Reaction of Erythrocytes

 

 

with AntiSerum with
Blood Antigens on Antibodies Anti-A Anti-B
Group Erythrocytes in Serum Antibody Antibody
0 None Anti-A - *
Anti-B
A A Anti-B + -
B B Anti-A - +
AB A,B None + I +

 

To determine an individual's blood group, a suSpen-
sion of the cells is mixed with a serum known to contain only
anti-A antibodies and one known to contain only anti-B anti—
bodies; it is then examined for agglutination after a few

minutes. The results are compared with the chart above.

24

From the above considerations the importance of deter-
mining the blood groups before performing a blood trans-
fusion is evident. The most important consideration is
generally believed to be the effect of the recipient's anti-
bodies on the cells of the donor, since the donor's anti-
bodies are diluted to the point where they fail to exert
any harmful influence on the recipient’s cells. The trans-

fusion possibilities found feasible are then:

 

 

 

 

 

onor
Recipien 0 A B AB
0 Yes No No No
A Yes Yes No No
B Yes No Yes Nog—
AB Yes Yes Yes Yes

 

 

 

 

 

 

 

In the period 1900-1940 or so, several important dis-
coveries were made: the inheritance of the ABO groups was
determined, some of the subgroups of ABO were discovered,
part of the ABO chemistry was unraveled, the blood groups
MN and P were discovered, and blood-bank techniques
continually improved. A new era in blood group work was
opened in 1939 with the discovery of the Rh blood groups
(to be described later).

We now summarize the present stage of knowledge. When
certain foreign substances (antigens) are injected into an
animal, there may appear in the blood serum after a varying

amount of time, substances (antibodies) able to react in a

 

25

specific manner with the antigen that elicited their pro-
duction. An animal that produces antibodies in response to

injection of antigen is called immunized. The presence of

 

antibody is detected by various reactions characteristic of
its interaction with the antigen. These reactions may occur
in vitroor in vivo.

A brief description of blood is necessary in order to
describe these processes in more detail. Blood is a body
constituent transporting food and oxygen as well as sub-
stances necessary for the defense of the cells against in-
vaders. The blood also removes waste products, and aids in
controlling the temperature and pH of the cells. About 5 to
10 per cent of the body weight, depending on the Species, is
made up of blood.

When blood to which anticoagulant is added is allowed
to settle or is gently centrifuged, it separates into solid
material and a supernatant yellowish fluid. The solid

material consists primarily of the erythrocytes (red cells),

 

leucocytes (white cells), and platelets. The supernatant

 

 

is called the plasma. When the plasma is allowed to clot,

part of it, called the fibrinogen, comes out as a fibrous

 

mass. The remaining material is called the EEEEE- Figure 3
is a schematic diagram showing the important components of
blood. The serum contains a number of simple inorganic and
organic compounds, and a large number of proteins.

Mammalian red cells are non-nucleated cells consisting

of a complex cell membrane with a "solution" of hemoglobin

 

 

 

//,

    

 

0 O .
o
. o

 

'\

/11111141/////////

[ii/I‘I7/1/If’frilll'

 

 

 

 

J
Figure 3 -

, PLASMA

26

Average per cent
of total blood
Proteins volume
Fibrinogen ‘

Albumins

Globulins >

atS'
ugars

Amino acids
Salts, eto.j

SERUM 5”

 

Buffy layer
Platelets
White cells (leucocytes)

FORMED
ELEMENTS

Red cells (erythrocytes) 45

The essential composition of blood.
.(After Cushing, reference 40.)

27

inside, and perhaps an internal structure called EIEEEE: The
hemoglobin makes possible the transport of oxygen to a degree
approximately 20 times that of mere solution of oxygen in
water. The nature of the orienting forces associated with
the hemoglobin molecules is unknown, but there is some
evidence that the interior of the red cell is in the liquid-
crystalline state (63). From evidence based on phase-
contrast and electron microscopg and fluorescent—antibody
techniques, the membrane seems to be the site of immuno.
logical activity. Yet the antigen-antibody reaction may be
very subtle, with the peculiar properties of the interior
playing an important role.

The manifestations of the antigen-antibody reactions

most important for our purposes are: (a) precipitation,
(b) agglutination, (c) combination with hapten, and (4)
lysis.

(a) When a soluble antigen is mixed with its apprOpri-
ate antiserum, a precipitate of antigen and anti—
body is formed. It is generally believed that
the precipitate is the result of a union between
the antigen and antibody molecules, the resulting
antigen-antibody complex separating from the
solution. Quantitative analysis of the reaction
may be made by a protein analysis of the pre-
cipitate.

(b) Agglutination, the main topic in this work, will

be treated in detail later.

28

(c) The combination of antibody with haptens is des-
cribed later in the section on antibodies.

(d) Lysis (breaking down of cells) is sometimes the
result of the combination of antigen and anti-
body. These substances alone do not cause lysis.

Additional components (viz., complement) in the

 

serum must be present. The conditions for lysis
are in general more complicated than for pre-
cipitation and agglutination. There are some
antigen-antibody reactions, resulting in agglutin—
ation in vitro, but causing lysis in vivo.. The
complement is normally present in serum, and is
not directly connected with the immunization
process.

The two general types of in vivo response are: (l)
hypersensitivity, an increase in the biological response to
the antigen; and (2) immunity, an increase in the resistance
to the injurious effects of the antigen. Hypersensitivity
is manifested as a shock reaction; immunity gives the
individual greater resistance to disease. In hemolytic
disease the cause is sometimes the presence of an antibody
specific for the individual's red-cell antigens. Although
a consideration of the physiological reSponse is valuable,it
appears somewhat outside the immediate scope of the present
work.

In the blood systems with which we shall be mainly

concerned, the antigenic material is believed to be on the

29

surface of the red cell. Antigens may be classified on
their biological and chemical behavior as: (1) complete (or

functional), and (2) incomplete (or haptenic). The complete

 

 

antigens are those which react in a specific way with anti-

bodies, and also induce antibody formation. The incomplete

 

antigens are those which do not elicit antibody formation
(unless attached to a protein), but will react in a specific
way with the antibody once it has been formed.

Antigens are substances of relatively high molecular
weight (most are proteins, some are muc0polysaccharides) and
their chemical arrangement is known in only a few cases.
Landsteiner (41) showed that, by coupling simple chemical sub-
stances to protein antigens, antibodies which reacted
specifically with the simple substance could be produced.
Landsteiner named these substances haptens. The specific
activity of the antibody with the hapten may be demonstrated
by testing the action of the antibody against the protein and
then against the protein with the hapten coupled to it. In
this way it becomes possible to observe reactions which are
due to the hapten alone. Since the hapten has a known
structure, whereas the protein usually does not, it is
possible to draw inferences about the nature of the forces
involved in the reaction.

At present the physical and chemical characteristics
that are necessary to make a substance antigenic are not
known in detail. Two generalizations may be made in regard

to: (1) molecular weight and complexity, and (2) biological

30

relationships. In general, good antigens have a molecular
weight of at least 10,000. Between molecular weights of
10,000 and 40,000 there is a fair correlation between the
ability of a substance to elicit antibodies and its molecular
weight. Above 40,000 the relationship breaks down. A cer-
tain degree of complexity also seems to be necessary. For
example, there are such proteins as gelatin which have mole—
cular weights of about 100,000 and yet are only weakly anti-
genic, presumably owing to their relatively simple chemical
structure. Normally an animal does not produce antibodies
against the constituents of its own body or to components
which ordinarily enter into the blood. It has been shown,
however, that if the proteins of an individual animal are
altered chemically and then injected into the animal, they
may elicit antibody production that causes the destruction
of normal tissue. In vitro measurements of the potency of
an antigen has the advantage that the variability inherent

in living animals is reduced.

Formation of Antibodies

 

Until recently, ideas as to the site of antibody
formation were only speculative. The antibodies are globu-
lins, and it was expected that those cells which produce
normal globulins also produce antibodies. Recently Nossal
and Mgkelg (43) demonstrated antibody formation by single
lymph-node cells incubated in microdrops. This finding

seems to be strong evidence that antibody formation occurs

31

in lymph cells. There is reason to suppose that antibody
formation also takes place in the reticuloendothelial
system. Electrophoretic-pattern results have identified
most antibodies with the gamma—globulin component of the
serum; only a few are identified with the beta-globulin.

The molecular weight of the antibody molecules is
approximately that of the normal globulin components. The
molecular-weight data are based on ultracentrifuge sedimen-
tation, light scattering, osmotic pressure, diffusion, and
viscosity. Boyd (38) cites a value of about 160,000 as
typical for man and rabbit, and the anti-pneumococcus
antibody in horse, pig, and cow is about 900,000. It is
generally believed that the serum proteins approximate
prolate spheroids.

The question now arises: how is the body able to pro-
duce antibodies? Several theories have been proposed (44).

We mention here the template theories and the selection

 

theories. Both types of theory assume that antibodies are
developed by a modification of the normal synthesis.

Among the most pOpular of the template theories is the
one proposed by Pauling. He supposes that the presence of
an antigen molecule at the site of globulin synthesis modi-
fies the normal sequence in which amino acids are put to-
gether to form the polypeptide chains of the globulin. The
modified structure of the protein molecule is such that it
is complementary to the antigen structure and thus can react

specifically with it. The chemical components of the

32

globulin and the sequence are supposed to be the same, but
the antigen influences the folding of the polypeptide chain.
The theory is simple and offers a lucid explanation of the
Specificity of antibodies. The essential distinguishing fea-
ture of this class of theories is that the information comes
from outside the cell. An objection to the template theories
is the persistence of antibody formation long after the anti-
gen has disappeared.

The selection theories, of more recent origin, have
been prepared by Jerne, Burnet, and Lederberg. One form of
the theory proposed by Jerne supposes that globulin mole—
cules of a very wide variety of configurations (and there—
fore of specificities) are continually produced by the body.
An antigen is presumed to combine with those molecules of
the correSponding Specificity. The antigen-globulin complex
is then phagocytized, transported to the antibody-forming
cells, and there dissociated. For some unSpecified reason,
the body is supposed to discard-the antigen, and to continue
to make more globulin molecules like those which combined
with the antigen. The entry into the circulation of the new

Specific forms constitutes the rise of antibody level.

In selection theories of antibody formation, the in-
formation as to the construction of the antibody is assumed
to exist within certain of the protein—forming cells in the
animal. The presence of an antigen serves merely to stimu-
late the proliferation of those cells Specific to it.

That is, a given antigen selects for multiplication the

33

clone of cells that can react with it. Each cell of the
clone knows how to make the Specific antibody even though
the complementary antigen may never have entered the body.
The primary motivation for acceptance of this theory is that
it can explain the differentiation between the self and the
non-self. Normally the body does not produce antibodies
against its own proteins, although evidently it can produce
antibodies against almost any foreign protein or certain
other complex molecules. The clonal-selection theory ex—
plans this lack of self-selsitivity as follows: during
embryonic life the immunological cells mutate frequently, and
produce all possible antibody patterns, some of which match
antigens native to the body. These antigens will kill cells
having the complementary pattern and leave only the cells
with antibody patterns correSponding to foreign antigens.
Later in life, a foreign antigen will stimulate Specific cells
to proliferate rapidly and produce the corresponding antibody.

The principal objection to this theory is the hugeness
of the information store that must be carried in the body.
But if the antigenic determinants-~the sites of specific
chemical activity-~are small enough, their number is not
inordinately large. Proponents of the theory estimate that
perhaps only 10,000 different patterns are needed.

At present, no experiment has been devised that re-
quires either theory to be rejected. It may turn out that
a complete explanation will eventually be provided by a more
general theory incorporating the mechanisms of both instruc-

tion and selection.

34

The Rh Blood Groups

 

In 1939 Levine and Stetson (45) reported the finding
of an unusual antibody in the serum of a woman (with a
history of several miscarriages) who had given birth to a
dead fetus. This antibody did not react with the cells of
the mother herself, but did with the cells of the father
as well as with those of approximately 80 per cent of
individuals in blood group O. Levine and Stetson inferred
that the fetus had inherited an antigen from the father, and
that the mother had become immunized by fetal red cells.

In 1940 Landsteiner and Wiener (46) immunized rabbits
and guinea pigs with blood cells from the rhesus monkey.
After removing species—~characteristic antibodies from the
serum of the rabbit-—they found an antibody which agglutin-
ated red cells of the rhesus monkey and also of 85 per cent
of white people tested in New York. Landsteiner and Wiener
named the new antibody anti-Rh, after the rhesus monkey.
Individuals whose cells were agglutinated by the new anti-

body were called Rh5positive. In that same year Wiener and

 

Peters (47) showed that this anti-Rh antibody was the same
as that which caused hemolytic reactions in patients who had
received repeated transfusions of ABO-compatible blood.

In 1941 Levine and his associates (48) found evidence
indicating the Rh incompatibility to be the cause of the

syndrome known as erythroblastosis fetalis. It became evi-

 

dent that this disease occurred in an Rh-negative mother

carrying an Rh-positive fetus. The Rh antigens of the fetus,

35

through placental transfer, caused the mother to produce
anti—Rh. More than one pregnancy is necessary to build up a
significant level of Rh—negative antibody. This antibody
passed back to the fetus and destroyed the fetal cells by
hemolysis.

By 1943, several types of Rh antibodies, designated as
anti—C, anti-D, anti-E, and anti-c were known. Fisher (37),
in a theory presented in 1943, assumed that since the actions
of anti—C and anti-c were antithetical, the genes and anti-
gens identified by these two antibodies were alleles. Two
other serums, anti—D and anti-E, which were available in
English laboratories at that time, were not antithetically
related to any known serum. In Fisher's theory each of the
six Rh antigens (four known, two proposed) was produced by
a single gene, and these occurred in three alleleic pairs:
D-d, C-c, and E—e. Another theory, due to Wiener, assumes
that a single gene with multiple alleles is involved in the
production in all of the six antigens. As the complexities
of the Rh system unfolded, Wiener postulated additional
genes. Moreover, Wiener, in 1946, challenged Fisher's
theory and stated that two additional factors, d and e, pre-
dicted by Fisher could not exist. Within a few years, how-
ever, the predicted factors were discovered. But Wiener
still did not accept the Fisher theory and the controversy

is still unsettled. In the words of Race and Sanger (1958):

36

The existence of three sites where Mendelian sub-
stitution can go on seems to us unassailable, and to
argue whether the three sites are to be placed with-
in or without the boundary of gene appears to be
particularly unprofitable at the present time when
no one seems to know what the boundaries of a gene
are.

Each of the two foregoing theories has its own system

of notation. The Fisher scheme leads to the Fisher-Race

 

system. The Wiener system is more complicated than the

 

Fisher-Race system, owing largely to its lack of theoretical
simplicity. For a thorough discussion of the anti-Rh
groups, Race and Sanger (37) should be consulted. Here is

an example of the terminologies:

 

 

Gene Combinations Rh Antigens

Fisher- Fisher-

Race Wiener Race Wiener
DCe R1 D RhO
DcE R2 C rh'
Dce RO E rh”
DCE RZ d HrO
dce r c hr‘
dCe R' e hr"
ch R"
dCE R

Y
Agglutination

 

The agglutination reaction is presumably no more than
the precipitation reaction occurring at the surface of the
particle, the antibody molecule forming a bridge between the

antigen molecules on different particles. Because the

37

particles give large volume relative to the antigen and anti-
body molecules; the reaction is effectively amplified on a
volume basis, as compared with direct precipitation. Hence
agglutination will produce a visible reaction at far lower
concentrations than will precipitation.

It is sometimes possible to extend the range of ag-
glutination tests by coating red cells or latex particles
with an antigen, and then mixing the cells or particles with
the appropriate antibody. Fluorescein-labeled antibodies
have been used in agglutination tests to locate specific
antigens on the surface of red cells, bacteria, and protozoa.
Under proper conditions, certain fluorescent aromatic iso-
cyanates will combine with the free amino-groups of protein
molecules without interfering with the Specificity of the
antibody. When the labeled antibody then combines with the
antigen, the site of the reaction can be identified by
fluorescent microscopy.

Masouredis (49), with 1131—1abeled anti-D, found that
homozygous DD cells bound about 1.6 times as much antibody
as did heterozygous Dd cells. He calculated the number of
combining sites to be about 6,400 for the heterozygous cells
and 10,300 for the homozygous cells. This ratio showed that
the D+ blood group antigens were heterogeneous and genetic-
ally determined.

Cohen and Zuelzer (50), by use of the fluorescent-
antibody technique, identified blood-group antigens in

erythrocytes. They were able to demonstrate the factors A,

38

B, and a variety of Rh antigens.

By hemagglutination-inhibition studies it is possible
to gain information about the nature of antigen. The in-
hibition of the usual agglutination reaction is a result of
the combination of a substance with the antibody in question.
The substance apparently combines with the antibody because
it is structurally similar to the original antigen. It is
possible to learn about the nature of the antigen by deter-
mining what type of substance will inhibit a particular
antigen-antibody reaction. By inference, one also obtains
information about the antibody molecule.

In the case of hemagglutination inhibition, a chemical
substance is added to a solution containing a known antibody.
Red blood cells carrying the correSponding antigen are added.
If the usual agglutination does not occur, then it is
assumed that the chemical, by virtue of its similarity to
the antigen, has combined with the antibody leaving less
free antibody to combine with the antigen on the red blood
cells.

The inhibition of anti-D, anti-C, anti-E, anti—c, and
anti-e by four ribonucleic acid derivatives was reported by
Hackel £2.2l- (51, 52). This work suggests that the Rh
antigens are at least partly nucleotide in nature. This sug-
gestion was given further support when Hackel and Smolker
(53) found that treatment of erythrocytes containing Rh anti-
gens by the enzyme ribonuclease lowered the agglutinability

of the cells. Their hypothesis was that if any of the

39

antigenic Specificity of the Rh system was due to ribonucleic
acid derivaties, then the enzyme ribonuclease should remove
them from the cell membrane. The hypothesis was supported
because the Rh antigens were affected and the others were

not.

There are antibodies (called incomplete or blocking)

 

which by themselves do not cause agglutination in saline,

but which have the same specificity as those antibodies which
do agglutinate red cells in saline. These incomplete anti-
bodies will agglutinate erythrocytes suspended in a protein
medium. The presence of incomplete antibodies is particul-
arly dangerous because it is not detected by ordinary aggluti-
nation or precipitation tests. A Special procedure, the

Coombs test, has been developed for the detection of these

 

antibodies. It is based on the fact that antibodies are
globulins which are almost identical with normal globulins.
Now, normal globulin--as well as antibody globulin--in
humans is, of course, antigenic in rabbits, for instance.
Antiserum from a rabbit immunized by injection with human
globulin will then react with human globulin, whether normal
globulin or globulin modified into antibody. Normal
globulin is not ordinarily attached to the surface of erythro-
cytes, whereas antibody globulin is. Hence, the presence

of an incomplete antibody in a human will cause attachment
of globulin molecules on the surface of erythrocytes; and
the presence of these globulin molecules will be manifested

through agglutination by globulin antiserum. The test then

40

consists of the following: erythrocytes to be tested are
separated from the serum and washed thoroughly in saline.
Anti-human globulin is added to the cells. Agglutination
occurs if the cells are coated with incomplete antibody; a
negative reaction indicates the absence of such an antibody.
Anti-human globulin is a specific reagent usually prepared

by immunizing rabbits.

Statement of the Problem

 

General Considerations Concerning the Effect of Magnetic
Fields on Biological Processes

As has already been stated, the homogeneous magnetic
field can only exert a torque on magnetic dipoles. It has
been shown that the action of this torque on individual iso-
lated atoms in material of biological interest is insignifi-
cant compared with thermal action. If there exists in Some
biological process an action which depends on mesomorphic
states, in particular, the nematic state, then it is possible
that the action could be influenced by magnetic fields. The
occurrence of the nematic form of the mesomorphic state, the
form which is readily influenced by magnetic fields, is a
question which seems to be practically uninvestigated.

We may also infer that the biological effects of
magnetic fields are probably subtle; else they would have
been discovered a long time ago. Hence, if we hope to de-

tect effects we must be prepared to either investigate very

41

sensitive biological processes or to develop Sensitive in—
strumentation.

In view of the fact that little is known about the
molecular structure, let alone function of the biologically
important molecules, we can only make hopeful guesses about
what processes should be studied. We should choose an ex-
periment which ideally has the following characteristics:
(1) it should be capable of yielding reproducible and un-
ambiguous results, (2) the experiment should be sensitive,
and (3) the process should be one which has been fairly well
studied so that normal behavior is easily recognizable.
With these ideas in mind, we chose the agglutination in the
Rh system as the object of study.

Specific Effects of Magnetic Fields in Agglutination,
in Particular on Human-Erythrocyte Agglutination

The agglutination reaction in human-blood systems is
one of the most intensively studied subjects in biological
science. With reSpect to the items (1), (2), and (3):

(l) Agglutination has the advantage that it is

. carried out in vitro, and thereby relatively
independent Sf—TETTEtionS in a particular
animal. _

(2) Agglutination, in particular, the Rh system,is

one of the most sensitive phenomena occurring
in serological reactions.

(3) The antigen-antibody Shows high specificity of

the type shown throughout the whole biological
world.

II. EVALUATION OF THE EFFECT OF MAGNETIC FIELDS ON

AGGLUTINATION: VISUAL SCORING

In the first experiments, the visual scoring technique,
as developed by Race and Sanger (37), was adopted because of

its simplicity and wide acceptance.

Materials and Procedures

 

Capillary blood was drawn from the fingers of D+ indi-
viduals. The blood was collected in a test tube filled with
normal saline solution (0.9%) and washed three times with
saline. The cells were then made into a 2 per cent
suSpension. The antiserum was serially diluted with saline
in factors of two, from full strength to a dilution of
1024. TenLA of the cell suSpension was added to 10A of
the antiserum (in various dilutions) in 6 x 50 mm test
tubes. The mixture was then incubated for various lengths
of time, first in an oven, but later in a water bath main-
tained at 37.5: 0.1 C. The work with the A, B, 0 system was
done at room temperature. Antisera from Knickerbocker
Biologicals, Inc. and the Ortho Pharmaceutical Corporation
were used.

At the end of the incubation time the mixture of cells
and antiserum was withdrawn from the test tube and gently
smeared on a microscopic slide. The test specimens incu—

bated in the magnetic field and the control specimens

42

43

incubated outside the magnetic field were put on the same

slide to facilitate comparison.

Magnetic Fields

 

Magnets

Permanent magnets.-—Alnico horseshoe magnets were

 

available from discarded magnetrons. Two magnets were
clamped with opposite poles facing, an iron block being
placed between one pair of faces, the samples being placed
in the gap between the other pair of faces. For homo-
geneous fields ferromagnetic material was excluded from the
gap. For inhomogeneous fields, suitably-shaped soft-iron
pole pieces were fastened to the faces of the gap. These
faces were l—inch by 3-inches rectangles, the gap being
typically l-inch. Under these conditions, the field was
about 3,000 gauss, with gradient less than 200 gauss/cm over
a region about 3/4 inch by 2-1/2 inches. By putting on a
wedge pole piece, the field could be increased to 7,000
gauss at the apex, falling to 3,000 gauss at the other side
of the gap, to give gradients of about 3,000 gauss/cm and
field strengths of about 4,000 gauss at the location of the
samples under study. The permanent magnets, along with the

controltube holder, are shown in Figure 4.

Electromagnets.--Two kinds of electromagnets were
available for the study. A pair of smaller ones (Model R3),

Modern and Classical Instruments Corporation, Livermore,

44

 

A“,

Figure 1|». The permanent-magnet fixture in the water bath. me 6 x 50 mm
test tubes used in the. tests are Shown in the magnetic field. The control
appears near the magnet.

45

 

 

Figure 5. The Model 11-8 electromagnet with the temperature fixture in the
field.

46

California, had l-l/2—inch circular pole faces and a gap ad-
justable from 0 up to 8 inches. The coils were watercooled,
and would set up 15,000 gauss in a 3/4-inch gap when fed
with 5 amperes at 100 V d-c. A larger electromagnet (Model
L128, Harvey-Wells Corporation, Framington, Massachusetts)
had 12-inch circular pole faces and a fixed 2-inch gap. Its
water-cooled coils set up a field of 12,000 gauss when fed
by 50 amperes at 100 V d-c. The electromagnets are shown in

Figures 5 and 6.

Visual Evaluation

 

Each pair of smears was examined by direct vision at
the heavier agglutinations and microscopically at the weaker
agglutinations. The observer rated each smear for its de-
gree of agglutination on the following scale originated by

Race and Sanger (37).

+++ = agglutination clearly visible to the naked eye
++ = very large agglutinates seen microscopically

+ = large agglutinates seen microscopically

(+) = smaller agglutinates seen microscopically

w = the smallest definite agglutinates

no agglutination and cells evenly distributed
These readings are scored by giving the reactions the follow-
ing values: +++ = 10, ++ = 8, + = 5, (+) = 3, w = 2, and
- = 0. To compare the agglutination in a serial dilution of

two samples, the scores are totaled and compared.

Field Mapping

 

Probes.--The field intensity B was measured by Hall-

effect instruments (Instrument Systems Corporation, Model A-

47

 

me Model L-128 electromagnet with test-tube holder in position.

The temperature-controlled water supply enters and leaves through the rubber

tubes shown .

Figure 6 .

.¢.ou=www cw cacao uaoaowamuum uocwma unocmEuoa onu mpfioww osu mo ooh:Om
oLH .poaonma mm :uwcouuw ucmumaoo mo wooH one mocwa unoucoo one .mownnum
:oHumcfiuDwam now mondu umou mo aOMunom ecu um paowm owuocwme onu «O on: - n enamwm

 

 

 

' I"!
II” n
duh:

 

 

 

48
H

 

 

 

 

 

 

 

 

 

49

102) and by flip-coil methods (Rawson, Model 720). The
instruments were checked against a reference magnet (Rawson,
Type 721) and against a nuclear-magnetic-resonance magneto-
meter. The probes were held at the end of a nonmagnetic
extension arm clamped to a milling-machine bed. The probes
could easily be positioned to 0.01 inch for surveying the
field. The gradient, dB/ds, in any given direction was ob-
tained by calculation. The fields were mapped for the vari-
ous geometries and magnetomotive forces used in the experi-

ments. Figures 7 and 8 show the results of a typical field

mapping.-

Test-Tube Holder

 

A fixture to hold the samples in the field of the 12-
inch magnet was constructed and is shown in Figure 9. The
temperature in the fixture was held at the desired point by
circulating water from a controlled bath through it. The
temperature gradient between any pair of tubes as determined
by thermocouple measurements was less than 0.1 C.-deg.

A similar fixture was constructed to keep the control
samples at the same temperature. The water bath fed the
fixtures in parallel so that the temperature difference
between them could be minimized. The difference was kept to

0.1 deg.-C.
Results

Visual Scoring

Effect of concentration.-—With anti-D (anti-Rh) an

 

L 4 l2 XIIC)

dB/dZ

8’

 

 

 

 

o 0.2 0.4 0.6 0-8
DISTANCE 2 FROM EDGE (in.)

flat
ppme

face

 

 

Figure 8 - Plot of magnetic field quantities as a function of distance 2

from apex of iron wedge placed in gap to produce inhomogeneous
field. The magnetic field intensity B determines the force on

permanent dipoles in the field; the product B dB/dz determines
the force on induced dipoles in the field.

L4
.

 

 

 

 

rubber hose

(In easurements in inches)

Figure 9 - The test-tube holder used to position the test tubes
in the field of the large electromagnet.

  
    

 

    
 
  
  
 

 

  
   
 

| l

   

 

 

[IITL'I'II 1"1'1111'

[i

 

  

 

 

 

 

 

 

EI'I

 

 

 

Figure 10 - Schematic drawing of the Coulter counter. The circled

insert shows the aperture through which the blood cells
are drawn.

A = aperture, S = saline, M = mercury, V = vacuum,
B breaker contacts, E = electrodes.

53

enhancement of agglutination in a magnetic field was observed.
Table 2 shows the results of a typical experiment with anti—D
serum and D—positive cells. At the highest concentrations,

no difference was scored, the agglutination being maximal on
the Race-Sanger scale even though greater clumping was ap-
parent for the sample incubated in the field. For inter-
mediate and low concentrations, a scoring difference is ob-
served. The overall results on the Race-Sanger scales give

a total score of 74 to 56, an enhancement of 32 per cent on

this arbitrary scheme.

Effect of field strength.-—Similar runs were made in

 

more or less homogeneous fields, the average strengths run—
ning from about 20 to 5,000 gauss. The results are Shown in
Table 3. When D-negative cells were incubated with anti-D
Serum, no reaction occurred at any of the field strengths used,
thereby indicating that the magnetic field does not produce
non-specific agglutination. Table 4 shows the effect of a

high magnetic field on the anti-D reaction.

Effect of antiserum type.--Similar results were ob-
tained with cells of other D-positive genotypes against anti-
D serum. Anti—C (anti—Rh') and anti-E (anti-Rh") sera
against appropriate positive cells yielded essentially the
same pattern as anti—D serum. Anti—c (anti—hr') and anti—

e (anti—hr") have not yet been tested because of the in-
creased complexity of the technique without commensurate in—

crease in information likely to be obtained.

54

TABLE 2

EFFECT OF ANTISERUM CONCENTRATION ON ANTI-D REACTION
WITH D-POSITIVE CELLS
(Field Strength = 2,000 Gauss)

 

 

Serum
Dilution Field Control

Neat +++ +++
2 +++ +++
4 +++ +++
8 +++ ++
16 +++ ++
32 ++ +
64 + <+>
128 + w
256 (t) -
512 (+5 -

Total Scores: 74 56

 

55

TABLE 3

ENHANCEMENT OF AGGLUTINATION BY MAGNETIC FIELDS
(Anti-D Serum, D-positive cells)

 

 

Field Strength Titration Scores Percent
Enhancement

(Gauss) Field Control in Field
-- 57 57 0
23 56 56 0
29 56 56 0
37 56 56 0
53 68 56 21
85 68 56 21
130 70 57 23
200 70 59 19
400 70 56 25
800 70 56 25
2000 74 56 32
3000 68 52 31

5000 74 57 30

 

56

TABLE 4

EFFECT OF MAGNETIC FIELD ON ANTI-D REACTION WITH
D+ CELLS
(Field Strength = 16,000 Gauss)

 

 

Serum
Dilution Control Field Control Field
Neat +++ +++ +++ +++
2 +++ +++ +++ +++
4 ++ +++ ++ +++
8 ++ ++ ++ ++
16 ++ +++ + ++
32 ++ ++ ++ ++
64 ++ ++ + ++
128 + ++ + ++
256 (+) ++ + ++
512 w ++ (+) +
1024 _ (+)
2048 - (+)
4096 . _ _£:)

70 88 67 92

 

57

When the antibodies of the ABO and MN systems were
tested in analogous experiments, no enhancement by magnetic
fields was observed under the conditions of our experiment
and our techniques of observation. These tests were not ex-
haustive, however, and we hesitate to claim definitely the
absence of effects. Under other conditions, agglutination
with these antibodies were, as reported by Foner (65), in-

creased by the magnetic field.

Effect of incubation period.--For antigen-antibody

 

reactions where enhancement is observed, the incubation
period at the normal temperature was varied by factors of
one-half and twice the normal period. As a rule, increasing
the period increased the degree of agglutinations slightly,
but definite quantitative relations have not yet been

established.

Effect of incubation temperature.--Changes of 1.5 C-

 

deg. above and below the normal temperature showed neglig-

ible effects.

Effect of field inhomogeneity.--In the experiments
described, the fields were only moderately homogeneous. To
see whether adventitious inhomogeneity was possibly the
source of this effect, strong inhomogeneity was introduced
by placing wedge-Shaped iron pole pieces over the magnet
faces. When the incubation tubes were placed at the apex
of the wedge, the enhancement appeared to be intensified.
Experiments to date, however, do not permit meaningful

quantitative evaluation of this effect.

III. EVALUATION OF AGGLUTINATION:

SEDIMENTATION TECHNIQUES

From Stokes' formula the terminal velocity of a small

sphere falling in a viscous fluid is:

v z % (D-d)g r2 = Cr2
7

where v is the rate of fall of the sphere; D and d are the
densities of the Sphere and the medium, respectively; g is
the gravitational constant; r is the radius of the Sphere;

7 the viscosity of the fuoid; and C a constant. For simpli-
city, we take erythrocytes, individual or aggregated, as
Spheres. Aggregates of cells will fall faster than indivi-
dual cells, and hence the rate of sedimentation may permit
an evaluation of the agglutination.

The effect of changed sedimentation rate by agglutinins
had long ago been noted. In fact, over twenty years ago,
Hirst and Pickels (54) in 1942 developed a photometric in-
strument to record the course of the settling.

In the present work, where the effect sought may be
small, the apparatus must permit Simultaneous observation
of samples within the field and out of the field. In our
laboratory such an apparatus has been designed and built,
but results are not yet available.

Sedimentation rate was measured in a simple electrical
manner by Schwan in 1948 (55). He measured the change in
electrical resistivity between two electrodes in a blood

58

59

suspension placed so that the resistivity changed as the
cells sank. He found that the resistivity could be Simply
related to the sedimentation rate. To use this change in
resistivity as a measure of the rate of agglutination, Schwan
measured sedimentation rate by observing the change in re-
sistance between electrodes placed vertically in the suspens-
ion. He was able to measure sedimentation rate with whole
blood (containing 40% cells). This method, however, is ap-
parently not feasible for our work with dilute suspensions
(2%) because the small change in resistivity is masked by
Spurious effects from the electrodes and the electronic equip-
ment. Our experiments have shown such small differences in

resistance that they cannot be meaningfully compared.

IV. EVALUATION OF THE EFFECT OF MAGNETIC FIELDSCflQ

AGGLUTINATION: PARTICLE SIZING

Introduction

 

By far the most extensive work on erythrocyte size
distribution was that of Price-Jones (56). By photomicro—
graphy of highly—diluted blood smears he obtained size
distribution curves for samples of very many kinds of blood.
He reported a normal distribution with respect to erythro-
cyte diameter with a mean of 7.202 microns and a standard
deviation of 0-172AL A distribution symmetric in diameter
would, of course, give a distribution in volume skewed to
the higher volumes. Price-Jones‘ procedure is time consum-
ing and does not give the distribution of cells as they
occur in a liquid medium.

Automatic instrumental methods giving the distribution
in liquid medium are thus highly desirable. The Coulter
counter, an electronic instrument developed for this pur-
pose,counts and sizes particles suspended in a conducting
liquid. The counter was invented by Coulter, U. S. Patent
No. 2,656,508, and described by him (57) and Brecher (57).
Its use for counting cells was reported in the references
just cited and for sizing by Mattern §£_gi.(58). Agglutin-
ation of platelets and erythrocyteswwas detected by Halloran

et a1. (59), and by Brecher et a1. (60), who used the size

60

61

distribution. Goodman (61) used the Coulter counter in

quantitative hemagglutination.

Description of the Coulter Counter and Particle-Size
Distribution Plotter

 

 

The instrument Operates on the difference in electrical
conductivity between particles in a suspended medium. A sus—
pension is pumped through a capillary aperture. The passage
of a non-conducting particle changes the conductance to an
extent dependent on the system. Accurate solution of the
problem Of the change in conductance is extremely difficult,
but to a first approximation the change is proportional to
the volume of the particle. When constant current is main-
tained through the aperture, the voltage is proportional to
the change in conductance. The pulses are then counted and
sorted. A schematic representation of the instrument is
shown in Figure 10.

In actual use a known volume of sample must be passed
through the orifice,preferably at a constant flow rate. The
liquid is forced through hydrostatically. In the present
experiments, 0.5 m1. is counted. A constant current passes
from known electrodes on the high_pressure side of the
aperture to a platinum electrode on the low—pressure side.
The polarity Of the electrodes can be varied at will,and it
is advisable to alternate the polarity between successive
runs to minimize polarization effects. In the case of
fragile particles, such as erythrocytes, it is best to use

as low a current density as possible in order to avoid

62

damaging the cells as they pass through the aperture, the
voltage pulses following amplification and shaping are fed
into a scaler. In the Model A counter the input thresh—
hold is variable and all pulses greater than a given height
are counted. In the Model B counter the upper limit as well
can be selected and all pulses with heights between the
lower and upper limit are counted. ‘With either model, the
size distribution is obtained by successfully varying the
limits on the scaler.

Obtaining the distribution can be made largely auto-
matic with the Model B by automatically setting the succes-
sive gate widths and recording the number of counts at each
setting by means of the Coulter plotter.

The particle size distribution plotter has 25
channels. A stepping switch changes the channels automatic-
ally in a sequence from lower to higher ranges. At each
step of the sequence a set of pulses is received and the
integreated output is fed to a recorder. A typical output
curve from the plotter is shown in Figure 11. With this
device, using the lOO—micron aperture, the 25-channel distri—

bution can be obtained in about 100 seconds.

63

IO AGGLUT HATIG

AGGLUTINATIOI

mm AMI-D

 

Figure 11. Volume distribution of agglutinated erythrocytes as
obtained with Coulter-counter plotter. Note the decrease in spike

height in the 7th spike(’Veiaglete). and the rise at about the 17th
spike (~doublets).

64

Choice of Aperture Size

 

‘The choice of aperture size is determined by the size
and concentration of the particles counted. The aperture
size selected should be small enough to give good resolution
and small coincidence rate, and large enough so that clog—
ging is negligible. Good resolution is obtained by making
the change in conductance relatively large; that is, by mak-
ing the volume Of the particles large with respect to
volume of the aperture Opening. ”Coincidences,” that is,
the occurrence of more than one particle at the same time
to give a pulse Simulating the passage of a single large
particle can be reduced by using a smaller aperture (or,
of course, by decreasing the concentration of particles in
the suspension with attendant increase in counting time or

statistical error).

The Aperture Current and Amplifier Settings
On the Model B there are controls for the variation
of the pulse amplifier and the current through the aperture.

Since the output of the electrodes is clamped regardless of

65

the current passing through the aperture, the pulse height
of a given particle will be prOportional to the aperture
current. For good resolution it is apparent that higher
current densities should be employed. The nominal current
through the aperture may be varied from 4 ma. to 0.0625 ma.
in factors of 1/2. With a 100-micron aperture, a nominal
current Of 1 ma. corresponds to about a current density of
12.7 amps/cm2. In the case Of erythrocytes, the current
density must be kept sufficiently low to avoid damaging the
cells. The amplifier settings can be used to increase the
pulse height, and an increase of a factor of two in the
amplification is approximately equivalent to halving the
aperture current. It is preferable to use the lowest
aperture current feasible in combination with the highest
amplification.

At high current densities we have found that the blood-
cell count is changed and we attribute this to Several ac-
companying factors: the heating of the fluid as it flows
through the aperture,and the distortion of the cells. The
aperture current should be large enough to give pulses high
above noise, but should not be SO great as to cause excess-
ive heating or disruption of aggregates.

Choice of amplification selection determines the size
range of the particles that can be counted and the pulse-
height resolution. An increase in amplification decreases
the range of particle sizes that can be counted and in-

creases the resolution available for the distribution.

66

Volume Distribution of Erythrocytes

 

Distribution in Normal Blood

 

Figure 11a shows the record made by the plotter of a
2 per cent solution of normal blood cells in saline. For
this record the setting of the counter amplifier was 1/2,
the control setting (reciprocal Of the aperture currenD was
2 , and the aperture diameter was 100 microns. The plot is
a histogram in which the spike height is proportional to the
number of particles whose volume lies between prescribed
limits indicated by the abscissa. Each pair Of limits de-
fines a ”window" or "gate" on the abscissa scale. The
first few peaks come from very small particles existing in
the saline or other dilutant alone. This debris is Of
little interest in the present work. At the fifth or sixth
peak a maximum in height occurs corresponding to a volume
of about 85 cubic microns attributable presumably to most
probable erythrocyte volume.

Later in the work a more precise determination Of the
volume distribution was obtained by use of a 50-micron
aperture and a modification of the size-distribution plotter.
The 50-micron aperture made possible a finer resolution of
particle size, since here an individual particle causes a
pulse large relative to that Obtained with a lOO-micron
aperture.

To see whether the data are under statistical control,
we made eleven replications of the actual count in each

window for a give blood sample. A Specimen of blood was

67
drawn, washed in saline, and made into a 2 per cent suspension
in 150 ml saline. From this same beaker ll successive size
distributions were Obtained at intervals of about 5 minutes,
each run requiring approximately 4 minutes. The data are
shown in Table 5. The suspension was stirred between runs,
but not during a run.

Inspection of the table, as well as formal statistical
analysis (chi—square), shows that the distribution in the
first two runs differs markedly from that in the last 9. To
see whether the last 9 were under statistical control, they
were analyzed in the following way. The averages for each of
the 17 windows (4 to 20) were computed for the 9 runs. These
averages were taken as the theoretical distribution. The
difference between the actual count and the average count
was then computed, with the results shown in Table 6. These
values were then summed to permit chi-square tests to be
made. The windows 4 and 5, presumably representing debris
or perhaps noise, were discarded in the summation.

By the usual rules, a chi—square test would require a
chi-square less than 149 at the 5 per cent level. Our value
Of more than 200 lies outside this,and by usual statistical
procedures we should not say that the data come from a single
distribution. At this stage we prefer to state that the pro-
cess is not under statistical control,and wish to refine our
technique and investigate our apparatus before we pursue the
statistics further.

The skewness of the size distribution shows then the
distribution is not normal. We seek a transformation of the

abscissa which may transform the distribution to a familiar

68

 

 

 

 

 

 

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70

one. One obvious transformation is the cube root of the
volume (corresponding to a distribution in cell linear
dimension); another is the logarithm of the volume (corre-
sponding to a distribution in the logarithm of either the
volume or a linear dimension).

It turns out that a better fit is provided by the
logarithmic transformation, as may be seen from Figures 12
and 13. Although the fit on log-probability paper appears
excellent, a chi—square test does not permit acceptance at
the 5 per cent level, largely because Of the discordance at
two adjacent channels; nevertheless, we accept the log-
normal as a working hypothesis while awaiting additional
data that will give a definite answer on this question.

Similar studies were made by Lushbaugh (62) who used
the output of the Coulter counter as input to a lOO-channel
pulse analyzer. In this way he was able to obtain very
accurate frequency distributions of erythrocyte volume.
Lushbaugh was able to distinguish normal human blood from ab-

normal cases by the fractional width of the volume distri-

 

bution, defined as the ratio of the distribution width at
half the mode, to the mode itself. To relate our findings

to those of Lushbaugh we have calculated the fractional

width of our lognormal distributions, and compared our values
with his (Appendix). A close agreement is obtained. We
infer that his findings,as well as ours, are consistent

with a lognormal distribution.

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73

Distribution in Agglutinated Blood

 

Figure llb shows a typical plot for a 2 per cent sus-
pension Of D+ blood incubated with 1:8 dilution anti-D serum
for 45 minutes at 37.50C. The first few peaks again repre—
sent dirt and debris that are of little interest in our work.
The maximum height occurring again at about 85 cubic microns,
presumably the most common erythrocyte volume, but the num-
ber is considerably smaller than in the unagglutinated blood.
At a volume of about double 85 cubic microns (specifically
peaks 13, 14, and 15) another maximum in the height appears.
Naturally we attribute this rise to agglutinated pairs of
cells. Triplets and quadruplets would be expected to occur,
but the resolution of the plotter is not high enough to de—
tect the occurrence.

The new distribution,being bimodah can no longer be
either normal or lognormal. The deviation from either law,
of course, may give a measure Of the effectiveness of the
agglutinin. It is likely that a superposition of two
simple distributions can approximate the actual distribution,
but we do not consider an exhaustive search on this question
worthwhile. Figure 14 shows the deviation from log normality
on a cumulative percentage plot.

Figure 15 shows the ratio of corresponding spike
heights, as a function of peak height for agglutinated blood
in comparison with normal blood. The decrease in singlets
is shown by the dropping of the curves below unity around
the peak 7, and the rise in the doublet peak appears as the

rising of the peaks around peak 13.

 

 

 

 

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blood, the plot is nearly a straight line, representing a
lognormal distribution. For the unagglutinated blood. the

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76

Choice Of a Statistic to Characterize Agglutination

 

Since the occurrence of agglutination depresses the
' total count in causing several particles to appear as a
single conglomerate, the total count serves as an approxi-
mate indicator Of the extent of the agglutination. We might
wish, however, to take advantage of the fact that formation
of doublets (or even triplets and quadruplets) would inde—
pendently serve to indicate occurrence Of agglutination. We
might suSpect, therefore, that the increase in height of the
"doublet peak" could be used in combination with the de-
crease in height Of the singlet peak to yield a statistic
of high sensitivity. Furthermore, the actual concentration
of erythrocytes is variable because of difficulties inherent
in the sampling procedure; hence, use of a ratio of peak
heights,which to first order should be independent of the
concentration,is desirable. Accordingly, we define a
statistic D/S intended to represent the ratio of doublet to
singlets, but spread out over the peaks adjacent to each
maximum in order to diminish statistical fluctuations:

D/S a (13 + 14 + 15)/(5 + o + 7).
In the same vein we could define a statistic (T/S), or
(Q/SL.intended to represent the ratio of triplets to sing-
lets Or quadruplets to singlets, etc. Most Of our work to
date has been concerned with the statistic (D/S). The
choice of statistic to characterize agglutination is, of
course, arbitrary; only experience will show what are the

more useful statistics.

77

Statistical Behavior Of (D/S)

 

To get some idea of the reliability of the statistic
(D/S), we investigate the statistical behavior on repli-
cation. When ten replications were made with 2 per cent sus-
pension Of freshly drawn unagglutinated blood, the coef-
ficient of variation (CV) of (D/S) value was about 10 per
cent. The coefficient of variation for the total count N
in the same experiment was 3 per cent. The coefficientCA?
for agglutinated blood is typically about 6 per ceng and for

N about 6 per cent.

TABLE 7

RELIABILITY OF (D/S)

 

 

 

Sample D/S

l 3.38

2 3.19

3 4.06 Incubation time = 30 min.
4 3.49 Temperature = 37.50C.

5 4.07 Conc. antiserum = 0.

6 3.98

7 3.52 Standard Deviation = .346
8 3.35 Coefficient of Variation
9 3.07 (CV) = 9.7%
10 3.70

Ave. 3.58

 

Dependence of D/S on Concentration C

 

Figure 16 shows the variation of the D/S value as a
function of the concentration for D-positive cells incubated
with anti-D for 45 minutes. Each point represents the aver—

age Of eight samples. The value ofDVSat zero concentration

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concentration-for D-positive cells incubated with anti-D
serum for 45 minutes at 37.50C.

79

is appreciably different from zero because the normal
distribution of normal blood appears to contain some cells
with a diameter double that of the average. There is also
the possibility that spurious coincidences due to simultane-
ous passage of cells through the aperture accounts for this.
The existence of rouleaux formation has to also be con-
sidered.

The value of D/S increases linearly from its value at
c = O to about c = 0.8 where it levels Off. The slope of
the curve in the linear region is about 50 per unit concen-
tration. The ratio of the upper limiting value of D/S to
the lower limiting value is about 5 and this is typical of
the spread Of this statistic over the range of concentration

zero tO unity.

Dependence of D/S on incubation period.--It is impor-
tant to establish the sensitivity of D/S to the incubation
period-~both to determine the timing tolerance for the in-
cubation and to get information bearing on the mechanism
of agglutination. Figure 17 shows D/S as a function of 't
for D-positive blood incubated at 37-1/2OC. with anti-D
serum for periods from 10 minutes to 90 minutes. The linear
dependence becomes almost a strict proportionality when the
residual value of D/S is subtracted, until a saturation at

very high concentrations occurs.

Dependence of D/S on incubation temperature T.--Just

as for incubation period 2:, it is important to establish the

80

 

 

 

 

 

O 50 IOO
INCUBATION PERIOD T011111.)

Figure 17 - Agglutination, as measured by D/S. as a function of incubation
time for D-positive cells incubated with anti-D serum at 37.50
at two concentrations.

81

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82

dependence of D/S on incubation temperature T. Figure 18
shows D/S as a function of concentration c for D-positive
blood incubated with anti—D serum for 45 minutes at 37.50C.
and at 36.7OC. The course of D/S with c is not signifi-

cantly different between the two curves.

Use of Q/S as a Measure of the Agglutination

The count of various windows representing singlets,‘
doublets, triplets, and quadruplets was taken as a function
of anti—D serum used to agglutinate D-positive cells incu—
bated for 45 minutes and shown in Figure 19. It is evident
that the ratio Of quadruplets to singlets seems to vary more
rapidly with concentration than either the ratio of triplets
to singlets of the statistic D/S. We call this the ratio of
quadruplets to singlets Q/S. Figure 20 shows Q/S plotted as
a function Of the concentration Of anti-D serum for cells
incubated for 45 minutes. The variation in Q/S from very
dilute solutiOns of antiserum to full strength is about 100
as compared to a typical variation of 5 in D/S for the same
range.

Figure 21 shows the variation of Q/S as a function of
time fOr incubation of various concentrations of anti-D.
It is interesting that the variation in time for this
statistic is not as great with D/S. The behavior of Q/S is
not as linear as D/S and, therefore, for testing of the
magnetic field effects, the latter was used. However,

further work on the use of Q/S is necessary.

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86

Correlation between Visual and Particle Sizing Measurements
of Agglutination

 

 

In two runs with anti-D serum we attempted to find a
relationship between the visual scoring of agglutination and
the D/S value from the distribution. The samples were pre-
pared in the usual way and incubated for 30 minutes at 37.50C.
Figure 22 shows the visual score plotted against the D/S
value. The D/S value over the range studied varies by a
factor of about 4, compared to a factor of 10 for the visual
score. These values would seem to indicate that the visual
scoring is more sensitive. A good correlation between the

visual score and the D/S could not be established.

Dependence of D/S on Magnetic Field Strength B

It is plausible that a strong and inhomogeneous mag-
netic field will be most likely to affect the agglutination
reaction. The field strength in the lZ-inch electromagnet
was brought up to B = 16,000 gauss and wedge-shaped pole
pieces were used to produce a gradient dB/dz = 1,500 gauss/
cm. Incubation took place at 37-1/20C. for one hour. To
see if there was a change in the distribution at a given con-
centration, we computed the ratio of the spike height for
each window in the distribution obtained for the test sample
incubated in the field, to that for the control sample incu—
bated outside the field. A plot of this ratio against
window number for various concentrations is shown in Figure
23. No meaningful trend can be detected, and we conclude
that the magnetic field has no effect on agglutination as

measured by the Coulter counter.

87

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To search for an effect over a wider range of vari-
ables, the behavior of the statistic D/S alone was investi-
gated. Run after run failed to disclose any difference be-
tween the test samples and the control samples. Figure 24,
for a typical run, shows D/S as a function of c for D-
positive cells incubated against anti-D serum for one hour

at 37—1/20C. No trend is evident.

91

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SIG.

V. DISCUSSION

Visual Method

 

The experiments in which agglutination was determined
visually show definite enhancement of agglutination by mag-
netic fields of moderate strength. Much more work needs to
be done to get quantitative expression of the enhancement,
and to see how field strength and field gradient affect the

agglutination at varying concentrations of antiserum, and

at varying periods and temperature of incubation. Until such

information is available, it will be difficult to postulate

mechanisms to explain the observed effect.

Counter Method

 

The experiments in which agglutination was determined
instrumentally, on the other hand, show no response to mag-
netic fields. If the counter completely failed to detect
any agglutination, it would, of course, be easy to assume
that in the counting procedure the aggregates are destroyed,
say by being torn apart when entering the aperture. But,
as has been established in the present work as well as that
of others, agglutination can be detected by the Coulter
counter and, in fact, there is promise that antibody titer

can be determined by it.

Reconciliation of Results

 

Although it may be premature to declare absolutely

92

93

that the discordance is not an artifact, we advance the
hypothesis that the effect in each case is real, but the
differences in methods of observation result in differences
in the kinds of aggregates observed. Specifically, we think
that in the visual technique the spreading of the suSpension
on the glass slide favors production of aggregates of two
or more erythrocytes lying flat side by side, the cell rims
being in contact over only a very small portion. Such
aggregates are easily noticable under the microscope. In
contrast, aggregates consisting of pairs of erythrocytes
with one lying flat above the other would not attract at-
tention in the visual observation, as the upper cell would
shield the lower one from view. In the Coulter counter
method, on the other hand, aggregates made up of cells ly-
ing in the same plane and touching over only a small portion
of the periphery would be easily destroyed, and counted as
singlets. The aggregates made up of erythrocytes sticking
firmly together on their flat sides are probably fairly
sturdy and would pass through as multiplets. In sum, the
aggregates discerned readily by the microscope are not de-
tected by the Coulter counter; whereas, those detected
readily by the Coulter counter are not easily discerned
under the microscope. Thus, it is possible that a given
method preferentially detects cells agglutinated in a given
manner.

If it can be substantiated that the two methods do in-

deed detect different manners of agglutination, then the

94

discordance between the two methods in fact gives us a new
tool for investigating the mechanism of agglutination. Un-
til then, we must admit that no effect of magnetic fields

of weak or moderate strength has yet proved itself discern—

ible by electronic counting methods.

Possible Mechanism for Effect of Magnetic Field on

 

Agglutination

 

There seems to be little diSpute that the magnitude of
the magnetic interaction energy u.B for a single atom is so
small that it will be swamped by thermal energy kT. There-
fore, any effect observed at room or body temperature must
be based on some sort of cooperative phenomenon (or else
some subtle statistical phenomena). Here the magnetic moments
are to be coupled in some way so as to have a resultant
moment giving an interaction energy large compared with
thermal energy. Mathematically speaking, the interaction
energy Np.B for N coupled atoms would be about N times that
for a single atom, while the thermal energy kT would remain
the same. Thus the value of about 10-3 for the ratio of
uB/kT for a single magneton at room temperature in a field
of 104 gauss could be increased to 10 or even 100 for a swarm

4 or 105 associated molecules.

of 10
The most common cooperative phenomena in biological
material are likely those concerned with the existence of

the liquid—crystalline or mesomorphic state of matter. It

 

 

is well known that magnetic fields can produce orientation

95

of associated groups of molecules in this state (see, e.g.,
references 26-28). Substances pass into the mesomorphic
state from the solid state by decrease of the long—range
binding energies relative to thermal disordering energy,
either by increase of temperature or by addition of solvents.
Substances pass from the mesomorphic state to the true (iso-
trOpic) liquid state by further increase of temperature or
further dilution with solvent. It is known that some bio-
logical materials exist in the mesomorphic state, in parti-
cular, certain erythrocytes (63, 64). Hence, it is not
fantastic that some component of human erythrocytes might
respond to moderately strong magnetic fields.

We suggest tentatively then that erythrocytes are
aligned and perhaps even displaced by the action of macro-
scopic magnetic fields. Such motion causes some active
antigen sites on the cell surface to take up positions favor-
able for reaction with antibody, free or bound on sites on
adjacent cells. In the case of the anti—D reaction, this
type of reaction produces a weak bond, readily disrupted by
any sort of mechanical action, such as stirring, smearing,
or rapid passage through a narrow orifice. Indeed, the
anti-D reaction has the reputation of needing experience and
skill to preserve the aggregates under the microscope. There
will, of course, be unstimulated agglutination of the same
type, as well as of the type where the flat sides of ad-
jacent discs are stuck together firmly. Hence the Coulter
counter would detect agglutination, but only that resulting

in formation of sturdy aggregates.

96

In summary, the salient features of the investigation
are the following:

1. The agglutination reaction with human erythrocytes
was studied for possible influence by static magnetic fields
of moderate strength. Agglutination as detected by visual
scoring was enhanced by inhomogeneous steady magnetic fields.
Agglutination as measured by the particle sizing methods
was not influenced by magnetic fields, homogeneous or inhomo-
geneous.

2. Particle sizing methods appear capable of furnish-
ing an instrumental method for quantitating agglutination,
but only for gross effects.

3. Evidently, differences in methods of observation
result in differences in the kind of aggregates observed.
Corroboration of such differences might furnish a basis
for reconciliation of the discordant results of magnetic
tests.

4. Extension of the agglutination studies where en-
hancement by magnetic fields is found seems worthwhile
undertaking for possible elucidation of agglutination
mechanism.

5. Extension of the magnetic studies on other immune
reactions should be undertaken to find whether the antibody
molecules themselves or the erythrocyte membrane are the

seat of the response to the magnetic fields.

10.
11.
12.

13.

14.

15.

16.

17.

18.

19.

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APPEND IX
Fractional Width of the Lognormal Distribution

The lognormal distribution is characterized by:
I 'Qflfl—Ut

dJll‘) ? € 20‘

44 VOW/TF7

where the median is taken as l,

 

 

and V is the standard devi-
-02
ation. The mode is at 6 The value of x at which the

ordinate is half the mode is given by the equation.

 

 

 

 

 

‘0L_______A_(1~) : {71' é§-;Ly
41.00::an M40191 8‘02) 0/
\ : @qx) | l Ep'q%;37rz: a
119W Q, 2 : O‘VETFWE.‘61 (NEW
- fist—‘83: s: a}
OY a 10.1. ._ t

6
a
(M541) +QG22332‘i—‘t 0“,; O
U St: “6" 5"" 62‘ View-(ct 961%??— -e‘G€r¢:Qm
-61 tfvahfl
“Avamdw1Jv%=Xid-=e (6 -€

Ax= ae :WCU‘WD

3"“ 3...). (I (770‘)
.935...— — 98-61M0J770‘) QM [/J770")

0M.) " ‘ —¢’- :
ax M e

 

This is the equivalent of Lushbaugh's (fl) calculated for the

lognormal distribution. Various values of (M w) for individuals

in our laboratory were computed using a lognormal distribution.
The results shown on the following page are in close agreement
to those of Lushbaugh.

101

o w. a: :> <4 x

6) EU H a) (n W

Mode
(windows)

9.0
8.2
9.2
10.2
9.4
8.6

102

.20
.26
.21
.18
.25
.25

(MM)
.48
.62
.60
.43
.59
.59

"I1111111111111“