TRANSFUSION

Vol. 16 No. 4
~~~

Scientific Articles

Use of a Low-Ionic-Strength Medium in Manual Tests for

Antibody Detection

H. C . MOOREA N D P. L. MOLLISON
From the M R C Experimental Haematology Unit. St. Mary’s Hospital Medical School. London, England

to warnings of nonspecific agglutination

The present observations confirm the value of suspend-
ing red blood cells in a low-ionic-strength medium in the
a t NaCl concentrations below 0.2 g/dl
first stage of the indirect antiglobulin test; that is, during
(0.03 M)7 and partly due to the observation
the period of incubation with antibody-containing serum.
The main advantage of this procedure i s to shorten the
that at low ionic strength, due presumably to
time of incubation. I n this respect a low-ionic-strength
aggregation of IgG, com plemen t com pon en t s
medium appears to be superior to albumin as a suspending
are bound to red blood cells,12leading to false
medium for the red blood cells. A further advantage is to
increase the uptake of certain antibodies; this effect was
positive results. l 1
pronounced with selected Rh antibodies believed to be of
low affinity.
Low and Me se te r g found that when red
blood cells were suspended in a solution of
T H E RATE OF ASSOCIATION of anti-D with sodium glycinate containing 0.03 M NaCI,
D-positive red blood cells can be increased buffered to pH 6.7 with phosphate, the in-
1,000-fold by lowering the ionic strength of cubation time of cells with antibody-contain-
the medium in which the reaction takes place ing serum could be reduced to five minutes in
performing
from 0.17 I to 0.03 I;’ see also Elliot, et ~ l . ~ the indirect antiglobulin test, and
In detecting a wide variety of blood group false positive results were of negligible im-
antibodies by the indirect antiglobulin test portance. The present work was undertaken
the use of a low-ionic-strength medium to investigate further the value of this
allows the incubation time to be reduced method and to define as closely as possible
from one hour to five minute$ moreover the the optimal conditions for enhancing results
titer of a wide range of antibodies is in- and avoiding false positive reactions.
~ r e a s e d .Although
~.~ a low-ionic-strength me-
Materials and Methods
dium has been used in automated blood
grouping tests,8 there has been much hesi- Low-Ionic-Strength Solution (LISS)’
tation in using this method in routine man- The solution described by L ~ and w MesseterO
ual tests for the detection of blood group as “LISS” consisted of:
antibodies. Reluctance to use a low- saline, 0.17 M, 180 ml
ionic-strength medium has been due partly phosphate buffer, 0.15 M, pH 6.7, 20 ml
and sodium glycinate, 0.3 M, pH 6.7, 800 ml.

Received for publication August 20, 1975; accepted In the present work, as sodium glycinate was
September 15, 1975. not available commercially, the solution was pre-
29 1
Transfusion Volume 16
July-Aug. 1976 Number 4
29 2 MOORE A N D MOLLISON
pared as follows: Glycine, 18 g was dissolved in ap-
proximately 500 ml distilled water and NaOH, 1.0
M, was added by drops, with stirring, until the pH
reached 6.7; (approximately 0.35 ml of 1.0 M
NaOH was needed). Twenty ml phosphate buffer,
0.15 M, pH 6.7, (made by adding approximately
equal volumes of 0.15 M N a 2 H P 0 4 and 0.15 M
NaH,PO,) was added, followed by 1.79 g NaCl
dissolved in approximately 100 ml distilled water.
The mixture was then made up to one liter with
distilled water. The conductivity of the final solu-
tion was found to be 3.7 mmho/cm a t 23 C.
Sucrose Low-Ionic-Strength Solution
This solution differed from "LISS" only in that
100 g sucrose was used in place of 18 g glycine.
Red Blood Cell Suspension
A concentration of 3 per cent washed red blood
cells was used instead of the 5 per cent recom-
mended by Low and M e s ~ e t e r .The
~ red blood
cells were always washed twice in saline before be-
ing washed once in LISS and then suspended in
LISS.
A n tibody-Containing Sera
One o r more examples of the following speci-
ficities were tested:
a) in agglutination tests: anti-A, -B, -I, -i, -PI,
-PP,Pk,-M, and -Lea;
b) in indirect antiglobulin tests, using an anti-
IgC serum: anti-D, -E, -C", -c, -e, -K, -Fy',
-Jk', -s, -Lub, -YP, -Csaand -Xga.
The Rh antibodies tested included examples
kindly supplied by Dr. B. E. Dodd. These anti-
bodies had been selected on the basis that they re-
acted well with enzyme-treated red blood cells but
only weakly or not a t all by the indirect antiglo-
bulin t e ~ t . ~ . ~
A reference preparation (68/419) of anti-D was
kindly made available to us by Dr. N. C . Hughes-
Jones. This preparation has a nominal value of 60
pg anti-D/ml and has been proposed as an inter-
national standard. I
c) In indirect antiglobulin tests, using an anti-
PIEor, occasionally, an anti-filclA serum:
anti-Lea, -Jka, Fy".
Antiglobulin Sera
The anti-IgG sera had been prepared in rabbits
or goats by injecting purified IgG obtained by
DEAE-cellulose chromatography. The sera had
an optimal dilution of approximately one in 250
with Rh-sensitized cells; anti-PIEand anti-PIclA*
*Hoechst Pharmaceuticals, Frankfurt, West
Germany.
Transfusion
July-Aug. 1976
were used a t a dilution of one in 50. In a few cases,
results were checked against a commercial anti-
globulin reagent.**
Indirect Antiglobulin Tests
One-stage test. One drop of serum o r diluted
serum was added to one drop of a 3 per cent sus-
pension of red blood cells in a small plastic test
tube and placed in a water bath a t 37 C. After
varying periods of incubation the cells were
washed four times. A f t e r discarding t h e
supernatant, two drops of diluted antiglobulin
serum were added and the tube spun immediately
at low speed for one minute. The deposit was then
gently resuspended and examined on a slide under
the low power of a microscope.
In tests with anti-complement-globulin sera,
since the sera containing blood group antibodies
had been stored a t 20 C for periods between one
half and 20 years, they were diluted in fresh serum
to provide active complement.
One-stage test with albumin. One volume of a 3
per cent suspension of cells in 0.147 M NaCl was
placed in a tube and centrifuged. After discarding
the supernatant, the cells were resuspended in two
volumes of 30 per cent albumin and three volumes
of serum or diluted serum were then added. After
incubation for periods between five and 30 minutes
the cells were washed four times in normal saline;
finally two drops of diluted antiglobulin were ad-
ded to the button of cells, mixed, centrifuged and
examined on a slide under the low power of a
microscope.
Two-stage test (modification of test described
by Polley and Mollison") One volume of serum
treated with four mg EDTA/ml was added to one
volume of a 3 per cent suspension of red blood
cells and incubated a t 37 C for five minutes. The
cells were washed four times. After discarding the
supernatant, one volume of fresh normal serum
was added to the button of cells and the mixture
incubated a t 37 C for five minutes. The cells were
then washed four times, the supernatant dis-
carded and two volumes of diluted anti-comple-
ment-globulin serum added. The mixture was then
centrifuged and examined on a slide under the low
power of a microscope.
Agglutination of Enzyme-Treated Cells
The method of Haber and Rosenfielde was used.
Agghtination of Untreated Red Blood Cells
One volume of serum was added to one volume
of a 3 per cent suspension of red blood cells and
**Ortho Diagnostics, Raritan, N.Y.
Volume 16
Number 4
LOW- ION IC-STRENGT H M ED1 U M 29 3
Table 1. Indirect Antiglobulin Tests (Anti-lgGI with Red Blood Cells Suspended in Saline
and LlSS Respectively

Incubation
Time Anti-D ( 1 in 100) Anti-c Anti-Xga
(min) Saline LlSS Saline LlSS Saline LlSS

1 %‘ 2 0 1 0 1
5 1 2 % 2% % 1%
10 2 3 % 3 - -
60 2 3% 3% 3 1% 2
90 2 3% - - 1% 2

“In this Table and in Table 2, the numbers indicate the intensity of the agglutination reactions on an arbitrary
scale of % to 4. (- = not done)

the mixture was left a t 37 C for five minutes be- Results

fore being lightly centrifuged. The button of cells
was gently resuspended and examined on a slide
under the low power of a microscope.
Titration of Antisera
Serial, doubling dilutions were made in a con-
ventional manner. When comparing results with
red blood cells suspended in saline and LlSS re-
spectively, the “master dilution” method was em-
ployed so that exactly the same dilutions were
used with cells suspended in saline and LISS re-
spectively.
Estimation of Drop Size
When a drop of fluid is delivered into a plastic
tube it is frequently noted that the drop is drawn
towards the wall of the tube and that this ap-
parently results in the delivery of drops of variable
size. To investigate this point, 51Crwas added to
serum and to a suspension of red blood cells in
saline and single drops were delivered into plastic
tubes which were then counted in a gamma
counter. The effect of delivering drops with the tip
of the pipette held close to the orifice of the tube
and at least one inch above the tube was investi-
gated, as was delivery into glass rather than
plastic tubes.
Indirect Antiglobulin Tests with Anti-IgC
With cells suspended in LISS, as compared
with cells suspended in saline, positive reactions
developed more rapidly and the antibody titer was
about four-fold greater. As Table I shows, after
one minute’s incubation positive results were ob-
tained with cells in LISS in all three examples, but
the results were negative with cells in saline in two
out of the three examples. With cells in LlSS the
reactions were maximal or almost maximal after
ten minutes’ incubation. After 90 minutes’ incuba-
tion the reactions with cells incubated in saline
were usually slightly weaker than those of cells in-
cubated in LISS for only ten minutes. However, in
some cases the maximum strength of the reaction
in the two media was similar. No obvious
difference was observed between the reactions
given by the anti-IgG serum and the commercial
“broad-spectrum” antiglobulin reagent. In tests
with the reference preparation the lowest con-
centration of anti-D which could be detected in the
indirect antiglobulin test after 15 minutes’ incuba-
tion using saline-suspended cells was 0.025 pg/ml
and using cells in LISS was 0.005 pg/ml.
The discrepancy between results obtained with
cells suspended in saline and LISS respectively

Table 2. Indirect Antiglobulin Tests (anti-IgG) with a ) Cells Suspended in Saline; bl Cells Suspended in 30%
Albumin and cl Cells Suspended in LlSS

Incubation
Time Anti-Jka Anti-Csa
(mid Saline Albumin LISS Saline Albumin L I ss.
~~ ~ ~

1 0 0 2 - - -
5 % 0 3 % % 1
15 % 1% 3 % 1 2
30 2 3 3 1 1 2
60 3 3% 3% 1 1 2
90 3% 3% 3% - - -
294 MOORE AND MOLLISON
was greater when using certain selected Rh anti-
sera. These sera had been selected (by Dr. B. E.
Dodd) because they gave very poor reactions in
the indirect antiglobulin test but strongly aggluti-
nated enzyme-treated cells. With some of these
antisera the indirect antiglobulin titer with cells in
LISS was almost as high as the agglutination titer
with enzyme-treated cells.
The effect of adding albumin to the mixture of
antibody-containing serum and cells was com-
pared with that of omitting the albumin but sus-
pending the cells in LISS. In all cases the degree
of augmentation of the reaction by suspending the
cells in LISS was far greater than that achieved
by adding albumin to cells in saline. Table 2 shows
an example. In case the combination of albumin
and LlSS should prove advantageous, 30 per cent
albumin was dialysed against LISS and then used
for the resuspension of red blood cells. One
volume of a 2 per cent suspension of cells in the
dialysed albumin was incubated with an equal
volume of serum and the cells tested in the usual
way. This method proved to be very insensitive.
Eflect of Washing Antibody-Coaled Cells in LISS
When cells were washed in LISS rather than in
saline before being tested with an antiglobulin
serum, only a very slight enhancement of the reac-
tions was observed; for example, in one series in
which eight different antibodies were tested, the
average “score” was 2.2 for cells washed in LlSS
compared with 2.0 for cells washed in saline.
Surprisingly, the effect of washing in LISS was
greater with cells which had been suspended in
saline during incubation with antibody, t h e
average score being 1.9 for cells washed in LlSS
compared with 1.3 for cells washed in saline.
Indirect Antiglobulin Tests Using A n t i - 8 1or
~ at which false positive reactions would occur when
Anti-PICIA
In one-stage tests with only five minutes’ incu-
bation of the mixture of cells, antibody and com-
plement, considerable enhancement of the reac-
tion was observed with cells suspended in LISS
rather than in saline. For example, in testing a
sample of anti-Fya, with cells suspended in saline
the titer of the antibody was two but with cells in
LISS was 32. Similar results were observed in
two-stage tests except that the reaction of cells in
LISS incubated with relatively high concentra-
tions of anti-Fya was apparently stronger than in
the one-stage tests.
Reactions with anti-Jka were similar. With one
example, in a two-stage test the reaction was
negative with saline-suspended cells, even when
the cells were incubated with the antibody for one
and a half hours in the first stageof the test. With
cells in LlSS the serum had a titer of eight even
Transfusion
July-Aug. 1976
when the period of incubation of cells with anti-
body was only five minutes.
Agglutination Tests with Untreated Red Blood
Cells Suspended in Saline or LISS
In general, the same results were obtained with
cells suspended in saline and LISS, titers being
either identical or differing by not more than one
doubling dilution. However, in testing examples of
anti-M it was consistently noted that the antibody
had a higher thermal range in tests with cells sus-
pended in LISS. For example, serum D.S. was
originally described by Cutbush and Mollison3 as
agglutinating MM cells in saline at 34 C but not a t
37 C and M N cells at 31 C but not a t 34 C. Tests
on a sample of serum stored since 1958 showed
that it had retained the same characteristics.
However, when the serum was tested with cells
suspended in LISS, positive reactions were found
with M M cells at 37 C and with M N cells a t 34 C .
On the other hand, the thermal range of examples
of anti-P,, -Lea, -I, and -i was no higher with cells
in LISS than with cells in saline.
Sizeof Drops Delivered from Pasteur Pipette
When delivered into glass tubes o r when de-
livered into plastic tubes from a height of at least
one inch, drops were usually within 10 per cent of
the mean value. However, when delivered into
plastic tubes with the tip of the pipette within a
centimeter o r so of the orifice of the tube the
volume delivered varied very widely. In one series
in which drops were delivered into eight consecu-
tive tubes, two drops were smaller than 6 jd and
two greater than 14 ~ l .
Conditions f o r the Development of False Positive
Results
In order to discover the concentration of NaCl
fresh blood was added to an excess of solution con-
taining NaCl and sodium glycinate, one volume of
blood was added to 20 ml of LISS and to a range
of solutions differing from LISS only in that the
concentration of NaCl ranged from 0.045 M to
0.025 M (in LISS the concentration is ap-
proximately 0.03 M). After incubation a t 37 C for
ten minutes the cells were washed and tested with
various antiglobulin sera. Reactions were only
doubtfully positive at 0.04 M NaCI, but at 0.035
M, positive results were regularly obtained with
anti-&, anti-PlEand with anti-IgC.
Evidently, when red blood cells suspended in
LISS are mixed with an equal volume of serum,
with an ionic strength equal to approximately 0.15
M NaCI, the final mixture must have an ionic
strength equivalent to about 0.09 M. Thus even if
two o r three drops of cells in LISS are inad-
vertently added to one drop of serum, there should
Volume 16
Number 4 LOW-IONIC-STRENGTH MEDIUM
be no risk of false positives due to too low an ionic
strength. I n order to check this conclusion,
measured volumes of cells in LISS and of fresh
serum were incubated together and tested with
various antiglobulin reagents. Provided that no
more than three volumes of cells in LISS were
added to one volume of serum no significant agglu-
tination by antiglobulin reagents was observed. On
the other hand, when testing stored red blood cells
it was noted that cells suspended in LISS tended
&,,.+
to give weak false positive reactions with anti-
Similar reactions were observed
in saline when tested with the same
cells
with
antiglobulin
serum and it was concluded that the use of a really
potent anti-plc serum is inadvisable in tests for
antibody detection. False positive results were not
a problem when using anti-IgC alone or when us-
ing a commercial “broad-spectrum” antiglobulin
reagent.
Discussion
T h e present observations confirm the
~ l a i mthat
~ . ~the use of sodium glycinate with
0.03 M NaCl as a suspending medium for red
blood cells in the indirect antiglobulin test en-
ables the incubation period to be very greatly
reduced. However, the present observations
suggest that rather than using five minutes as
previously s ~ g g e s t e d ,it~ might be wiser to
use ten minutes since in several instances the
reactions were found to be stronger at ten
minutes than at five minutes (Table I).
Previous authors7egseem to have dealt only
with anti-IgG but our observations show that
anti-PIE and anti-PIclAreactions a r e also
usually optimal after only five minutes’ in-
cubation, either in one stage tests in which
fresh serum is incubated with cells in LISS or
in the two-stage test in which EDTA-treated
serum is first incubated with cells in LISS for
five minutes and then (after washing) the
cells are resuspended in saline and incubated
with fresh serum for a further five minutes
before being finally washed and tested with
antiglobulin serum.
The present observations show that some
cold agglutinins can be detected at higher
temperatures when reacted with red blood
cells in LISS; only examples with specificity
anti-M were found to behave in this way.
Elliot, et ~ lpreviously
. ~ noted that agglutina-
29 5
tion by anti-M but not by antibodies of the
ABO and Lewis systems was enhanced by
the use of a low-ionic strength medium.
The findings with the selected Rh anti-
bodies provided by Dr. B. E. Dodd, as exam-
ples which agglutinated enzyme-treated red
blood cells but reacted very weakly or not at
all in the indirect antiglobulin test, are of spe-
cial interest. When these sera were tested
against red blood cells in LISS the indirect
antiglobulin titer was sometimes almost as
high as the titer with enzyme-treated cells al-
though, as described by Casey, et ~ 1 the . ~
reactions in the indirect antiglobulin test with
saline-suspended cells were very weak. These
observations strongly support the suggestion’
that these antibodies are of low affinity.
It is important that equal volumes of cells
in LISS and of serum should be incubated to-
gether. Although the addition of a consider-
able excess of LISS would be needed to-pro-
duce false positive reactions due to too low an
ionic strength in the medium, the addition of
an excess of serum, by raising the ionic
strength, would diminish sensitivity. When
using glass tubes it is satisfactory to deliver
the serum and cells as drops from an or-
dinary Pasteur pipette. However, with plastic
tubes which attract drops by electrostatic
force the volume of drops varies too widely,
as demonstrated in the present work. With
such tubes it is therefore recommended that
some method, such as the use of an Eppen-
dorf pipette, which ensures delivery of a fixed
volume (e.g. 50 PI) should be employed.
It is worth emphasizing a few practical
points in the performance of the test: 1) It is
essential to wash red blood cells in saline be-
fore suspending them in LISS as otherwise
false positive results may be obtained; 2)
LISS should be stored at 4 C and, unless pre-
viously sterilized, should be discarded after a
few days as it appears to be a good medium
for the growth of microorganisms. It is con-
venient to autoclave aliquots of LISS so as to
have a sterile supply of solution available for
each day’s use. 3) When diluting anti-sera
before testing, the diluent should be 8.5 g/1
NaCl buffered to pH 6.7. The same solution

MOORE A N D MOLLISON
should be used for washing cells prior to
resuspending them in LISS.
There would appear to be considerable ad-
vantages in using cells suspended in LISS in
the routine performance of the indirect anti-
globulin test. With short periods of incuba-
tion (e.g. 10 minutes) the method is more
sensitive than that of suspending red blood
cells in albumin and the sensitivity is on the
average slightly greater than that achieved
by prolonged incubation (90 minutes or
more) with saline-suspended cells. With
certain antibodies, presumably those of low
affinity, the increased sensitivity using red
blood cells in LISS compared with red blood
cells in saline is very striking.
References
I.Bangham, D. R., H. H. Gunson, and N. C.
Hughes-Jones: To be published.
2. Casey, F. M., B. E. Dodd, and P. J. Lincoln: A
study of the characteristics of certain Rh anti-
bodies preferentially detectable by enzyme tech-
nique. Vox Sang. 23:493, 1972.
3. Cutbush, M., and P. L. Mollison: Relation between
characteristics of blood-group antibodies in vifro
and associated patterns of red cell destruction in
v i v a Br. J. Haematol. 4:115, 1958.
4. Dodd, B. E., and D. A. Eeles: Rh antibodies de-
tectable only by enzyme technique. Immunology
4:337, 1961.
5. Elliot, M. E., Bossom, M. E. Dupuy, and S. P.
Masouredis: Effect of ionic strength on the
Transfusion
July-Aug. 1976
serologic behavior of red cell isoantibodies. Vox
Sang. 9:396, 1964.
6. Haber, G, and R. E. Rosenfield: Ficin treated red
cells for hemagglutination studies. In: P. H.
Andresen-Papers in Dedication of his 60th Birth-
day, Munksgaard, Copenhagen, 1957, p 45.
7. Hughes-Jones, N. C., B. Gardner, and R. Telford:
The Effect of pH and ionic strength on the reac-
tion between anti-D and erythrocytes. Immu-
nology 7:72, 1964.
8. Lalezari, P.: New method for detection of red
blood cell antibodies. Transfusion 8:372, 1968.
9. Low, B., and L. Messeter: Antiglobulin test in low-
ionic strength salt solution for rapid antibody
screening and cross-matching. Vox Sang. 26:53,
1974.
10. Medical Research Council: Production of antibody
against purified yG-globulin in rabbits, goats,
sheep and horses (Report of a Committee). Im-
munology 10:271, 1966.
11. Mollison, P. L.: Blood Transfusion in Clinical
Medicine, 4th Ed. Oxford, Blackwell Scientific
Publications, 1967, p. 439.
12. -, and M. J. Polley: Uptakeofy-globulin and
complement by red cells exposed to serum a t low
ionic strength. Nature 203:535, 1964.
13. Polley, M. J., and P. L. Mollison: The role of com-
plement in the detection of blood group anti-
bodies: Special reference to the antiglobulin test.
Transfusion 1:9, 1961.
H. C . Moore, M.D., Baptist Memorial Hospital, 899
Madison Avenue, Memphis, Tennessee 38103.
P. L. Mollison, M.D., M R C Experimental Haematol-
ogy Unit, St. Mary’s Hospital Medical School, London
W21PG, England.