Vox Sanguinis (2002) 83, 63– 69

© 2002 Blackwell Science

ORIGINAL PAPER

A novel method using formamide for the elution of

Blackwell Science, Ltd

antibodies from erythrocytes

L. Caruccio, K. Byrne, J. Procter & D. Stroncek
National Institutes of Health, Warren Grant Magnuson Clinical Center, Department of Transfusion Medicine, Bethesda Maryland, USA

Background and Objectives Accurate identification of antibodies that sensitize red

Received: 30 October 2001,
revised 22 February 2002,
accepted 25 February 2002
blood cells (RBCs) involves dissociating them from RBCs using an in vitro elution
method that does not alter their antigen-binding properties, and analysis of the eluates
against a panel of RBCs.
Materials and Methods A method was developed that allowed efficient RBC anti-
body elution. Human polyclonal anti-D was used to sensitize Rh-positive RBCs, and
known antigen–antibody disruptive reagents were tested using these RBCs. The best
reagent conditions were optimized. Eluates made were tested and compared to results
obtained with a glycine-acid-based commercial elution kit to determine efficacy. Patient
samples that were positive with direct antiglobulin tests (DATs), and in vitro commer-
cial antisera-sensitized RBCs representing clinically significant antibodies, were used
for evaluating the new method.
Results The formamide method was efficient at removing antibodies from RBCs. The
patient samples with a positive DAT had antibodies recovered with the same specifi-
city when compared to the acid-based technique. The length of preparation time was
similar for both formamide and acid-based methods. Results of testing the eluates made
from reagent RBCs sensitized with commercial antisera were distinct with antigen-
positive and -negative erythrocytes.
Conclusions The formamide method compares well with acid techniques and may be
an alternative choice of elution method.
Key words: DAT, elution, formamide sensitization, RBC.

in patient diagnosis and treatment. In order to identify the

Introduction
In transfusion medicine it may be necessary to remove anti-
bodies (Abs) that have sensitized red blood cells (RBCs) in vivo
when evaluating a patient’s positive direct antiglobulin test
(DAT) [1,2]. There are many reasons why patients can present
with a positive DAT and these include haemolytic transfusion
reactions, autoimmune haemolytic anaemia, drug-induced
haemolysis, and haemolytic disease of the newborn (HDN)
[1]. The identification of Abs that cause positive DATs can aid
Correspondence: Lorraine Caruccio, MT(ASCP), SBB, PhD, National
Institutes of Health, Warren Grant Magnuson Clinical Center,
Department of Transfusion Medicine, Bethesda, MD, USA
E-mail: lcaruccio@mail.cc.nih.gov
attached Abs it is necessary to remove and test them against
a panel of RBCs of known phenotypes. Elution, the technique
of removing and recovering bound Abs, was first pioneered
by Landsteiner & Miller [3]. Since then, several different
methods have been developed and used with various degrees
of success.
RBC antibody-elution methodologies use various disruptive
agents and conditions such as heat (56 °C), freezing, acidifica-
tion, sonication, chaotropic ions and organic solvents (ether,
toluene, dichloromethane) [4–9]. Each elution reagent and
condition has been somewhat successful; however, no one
method is superior. Different types of antigens are present on
the surface of RBCs or integrated through the RBC membrane
and the specific biochemical nature of these antigens may
be important with regard to the efficiency with which

63
64 L. Caruccio et al.
antibodies can be removed and recovered. No single method
will successfully elute all antibodies from RBCs and this includes
the commercially available kits which are based on the dis-
ruption of antibody–antigen complexes by acidic reagents
followed by neutralization with a basic buffer. Although
commercial kits have been in use for many years, the acid
required to elute antibodies can be potentially degrading to
the antibodies recovered, especially those of the Kell blood
group system, and may limit their storage conditions. Also,
potentially dangerous preservative reagents such as sodium
azide are necessary to avoid microbial contamination in
commercial acid-based kits, and this has added health risks
and dangers to the technologists using them.
The purpose of this study was to develop a more universal
and efficient method of preparing eluates that is easy to
perform, requires minimal preparation time, is low cost, has
good antibody recovery and can be used with limited amounts
of sample.
Materials and methods
Study design
Several new, potentially useful elution reagents were com-
pared. They were tested using Rh-positive anti-D in vitro-
sensitized RBCs. Of the reagents tested, formamide was
found to be the best as compared to results with a commercial
acid-based kit. Elution with formamide was optimized and
its efficacy assessed using RBCs from patients with positive
DATs, umbilical cord blood and RBCs sensitized in vitro with
reference antisera. Eluates were tested in both the micro-
column and tube agglutination antibody-detection assays.
Other, older reagents (such as xylene and ether) were not
used as they are highly toxic and hazardous, and their use is
currently avoided in most clinical laboratories.
Blood samples
Peripheral blood was obtained from patient samples at the
Warren G. Magnuson Clinical Center (Bethesda, MD) and at
INOVA Hospital (Fairfax, VA). Umbilical cord blood samples
from group A or B neonates born to group O mothers and
suspected of having ABO HDN were obtained from INOVA
Hospital.
Elution reagents
The reagents tested included those known to disrupt antibody–
antigen binding reactions in vitro. These included an organic
base (ethanolamine, 50 and 100 mM); a highly concentrated
salt solution (MgCl2, 1 and 3 M); hydrodynamic agents (ethylene
glycol and formamide (1·0 g/ml)); an organic polycation
(putrescine, 1 M); a basic detergent [sodium dodecyl sulphate
(NaOH–SDS), 0·1 N to 0·1%]; and a chaotropic agent
[potassium thiocyanate (KSCN), 1 and 3 M]. All reagents
except formamide were purchased from Sigma Chemical Co.
(St Louis, MO). Deionized formamide was purchased from
Amresco (Solon, OH).
Preparing acid eluates
Acid eluates were prepared using commercial kits (Elu-
KitII, Gamma Biologicals, Houston, TX; and R-E-S kit,
Dominion Biological Ltd, Nova Scotia, Canada) following
the manufacturers’ instructions with some modifications.
Saline was used instead of the commercial wash solution to
maintain consistency between the different elution methods
and to avoid false-positive results [10]. The commercial kit
instructions also recommended using 20 drops of RBCs to
prepare an eluate; however, it was not always possible to do
so as some sample volumes were limited.
Preparing formamide eluates
To prepare formamide eluates, 5–20 drops of red cells were
transferred to a 12 × 75 mm glass test tube and an equal
volume of saline was added. The cells and saline were mixed
and an equal volume of formamide was added and mixed
using a Pasteur pipette. The mixture was centrifuged at
≈ 800 g for 5 min. The supernatant was then transferred to a
new test tube and diluted 1 : 2 with saline. Saline and super-
natant were mixed using a Pasteur pipette and centrifuged at
800 g for 5 min. The supernatant was the eluate. If the eluate
was too viscous, it was filtered through a 0·22-µm syringe
filter (Milex-GV; Millipore, Molsheim, France), centrifuged a
second time, or passed through a G-25 Sephadex spin column
equilibrated with physiological saline (Sigma Chemical Co.).
When eluates were tested by tube-agglutination assays, two
or three drops of either albumin or low-ionic-strength solution
(LISS) were used in order to overcome the disruptive effects
of formamide on antigen–antibody binding.
In vitro RBC sensitization with reagent antisera
To determine whether the formamide method would elute
Abs specific to some of the most clinically significant blood
groups, antigen-positive RBCs were sensitized using human
and non-human commercial polyclonal and monoclonal anti-
sera. Monoclonal commercial antisera were tested to identify
possible applications for industrial laboratories required to
isolate monoclonal immunoglobulin M (IgM) class antibodies
for preparation of antibody reagents. This could have future
applications in research and industry.
RBCs were washed three to five times with physiological
saline, or until no haemolysis was present. Equal volumes of
antisera were added to equal volumes of washed packed
© 2002 Blackwell Science Ltd. Vox Sanguinis (2002) 83, 63–69
 Formamide elution of antibodies 65

RBCs, and the tubes were sealed with parafilm and placed in
a water bath at 37 °C with agitation for 2–3 h. After incuba-
tion, all tubes were centrifuged at 800 g for 5 min and the
antisera supernatant was discarded. Sensitized RBCs were
washed repeatedly (typically three to five washes) with
physiological saline until the supernatant was clear and no
residual Abs were detected in the last wash.
Commercial human source, polyclonal antisera were used to
sensitize included Rh (C, E, c, e), Kell (K, k), Kidd (Jka and Jkb),
MNSs (s) and Duffy (Fya and Fyb). The polyclonal C, E, e, K,
k, Fya and Fyb antisera were from Ortho Clinical Diagnostics
(Raritan, NJ), polyclonal Jka antisera was from Immuncor
(Norcross, GA), polyclonal Jkb was from BCA (West Chester,
PA) and the polyclonal antisera for s was from Gamma
Biologicals. Monoclonal antisera used were specific for ABO
(A,B), Rh (C, E, c, e) and Kidd (Jkb), and were purchased from
Ortho Clinical Diagnostics.
Confirming antibody sensitization and
testing of eluates
Sensitization of all RBC samples was confirmed by both tube
agglutination and gel microcolumn DATs. Cell suspensions
(3–5%) were washed three times with physiological saline
and decanted to a dry cell button. Antiglobulin reagent
(polyclonal, IgG specific, complement specific, or saline as a
negative control) was added to the button. Tubes were cen-
trifuged for 15 seconds at 800 g and read according to the
manufacturers’ instructions. Gel microcolumn DATs were
performed using the ID-MTS system (Micro Typing Systems,
Pompano Beach, FL) [11]. Fifty microlitres of a washed 0·8–
1·0% cell suspension was added to an IgG gel card. Gel cards
were centrifuged for 10 min at 90 g in an MTS centrifuge (Ortho
Diagnostic Systems, Inc.) and analysed macroscopically.
Eluates prepared using either formamide or acid were tested
for specificity using the IgG-gel microcolumn assay (Ortho
Diagnostic Systems, Inc.) and the tube assay. In the gel assay,
25 µl of eluate was added to 50 µl of 0·8% cell suspensions of
screening cells, A1 and B cells. Cards were incubated at 37 °C
for 15 min, spun for 10 min at 90 g in an MTS centrifuge
(Ortho Clinical Diagnostics) and read macroscopically.
The instructions recommended by the manufacturers of the
elution kits were followed when testing eluates in the tubes,
except that saline was used in place of working wash solution
(Gamma Biologicals and Dominion Biologicals Ltd).
Results
Selection of the most promising elution method
Several reagents were tested for their ability to elute anti-D from
RBCs that were sensitized in vitro. As expected, glycine-acid
eluted anti-D. Eluates made using potassium thiocyanide and
ethanolamine reacted with all cells tested. Formamide eluted
anti-D specifically (Table 1). No anti-D could be detected in
eluates that were prepared with magnesium choride, putrescine,
ethylene glycol, or SDS. All reagents were tested at 4 °C,
room temperature and 37 °C. Results were found not to be
affected by temperature. All reagents except glycine-acid in
the commercial kit caused some RBC lysis and haemoglobin-
stained eluates. The results with formamide were sufficiently
promising to warrant further study.

Table 1 Gel-agglutination assay results of

eluates prepared using various reagents Results Modification
Reagent Concentration Cell 1,2,3a 4 °C, RT, 37 °C Haemolysis

KSCN 1 and 3 M 1+ for all 1+ for all Yes

MgCl2 1 and 3 M 0 for all 0 for all Yes
Ethanolamine 50 mM 1+ for all 1+ for all Yes
100 mM 1+ for all 1+ for all Yes
Putrescine 1M 0 for all 0 for all Yes
Ethylene glycol 1 g/ml 0 for all 0 for all Yes
NaOH, SDS 0·1 N, 0·1% 0 for all 0 for all Yes
Formamide 1 g/ml 2+ for Cell 1,2 2+ for Cell 1,2 Yes
(deionized) 0 for Cell 3 0 for Cell 3
Commercial kit Unknown 2+ for Cell 1,2 2+ for Cell 1,2 No
(glycine-acid) 0 for Cell 3 0 for Cell 3

a
Eluates were tested against screening cells (Cell 1, Cell 2, Cell 3). The eluates were prepared from
Rh-D-positive cells sensitized in vitro with anti-D.
Cell 1 was D-antigen positive; Cell 2 was D-antigen positive; Cell 3 was D-antigen negative.
Values represent the reaction grade of testing.
RT, room temperature.

© 2002 Blackwell Science Ltd. Vox Sanguinis (2002) 83, 63 –69

66 L. Caruccio et al.

Table 2 Acid and formamide elutates prepared from nine of 25 patient samples differed when tested in the gel-agglutination assay

Sample Gel-DAT Cell 1 Cell 2 Cell 3 A1 Cell B Cell

numbers results acid/formamide acid/formamide acid/formamide acid/formamide acid/formamide

5 3+ 4+/4+ 4+/4+ 0/3+ 0/0 0/0

9 3+ 2+/4+ 2+/4+ 3+/4+ 3+/4+ 3+/4+
11 2+ 0/4+ 0/4+ 0/3+ 1+/3+ 2+/4+
12 2+ 0/3+ 0/3+ 0/3+ 1+/3+ 2+/3+
14 1+ 3+/4+ 2+/4+ 2+/4+ 3+/4+ 4+/4+
15 1+ 0/2+ 0/1+ 1+/1+ 0/1+ 0/1+
16 1+ 2+/2+ 2+/2+ 0/2+ 2+/4+ 3+/2+
18 1+ 0/3+ 0/3+ 0/3+ 1+/3+ 1+/3+
19 3+ 2+/3+ 1+/3+ 2+/3+ 1+/3+ 1+/3+

Eluates were prepared from patient samples using a commercial acid kit and the formamide method and tested against a three-cell screening panel (Cell 1,
Cell 2, Cell 3), and A1 and B cells.

cells in the tube agglutination assay. Insufficient sample was

Optimizing formamide elution
As similar quantities of anti-D were eluted by formamide at
4 °C, room temperature and 37 °C, all studies were performed
at room temperature. Three formamide incubation periods
were tested: 0 min, 5 min and 10 min. The quantity of anti-
D eluted when RBCs were centrifuged immediately after
adding formamide (no incubation) was the same as when RBCs
were incubated with formamide for 5 min or 10 min before
centrifugation. Three different formamide concentrations
were also tested. The ability of formamide to elute anti-D was
compared using undiluted reagent (1·0 g/ml) and reagent
diluted with physiological saline at a ratio of 1 : 2 or 1 : 4.
There was no improvement of antibody recovery with diluted
formamide. All further testing therefore used formamide at
a concentration of 1·0 g/ml. Testing was performed at room
temperature and RBCs were centrifuged immediately after
formamide was added.
Eluting antibodies from patient samples
Both gel and tube DATs were performed on 25 samples obtained
from patients. Twenty-two of 25 samples had positive DATs
by both methods. Acid and formamide eluates were made
from the three DAT-negative samples and these eluates were
non-reactive in both the gel and tube agglutination assays.
Eluates made from the 22 DAT-positive samples demon-
strated various patterns of reactivity. Nineteen of the 22
eluates were reactive in the gel assay. In 10 of the 19 samples
the grades of the gel reactions of the acid and formamide
eluates with all five panel cells (screening cells 1,2,3 A1 and
B) were the same or differed by only one grade. However, gel
assay reactions of formamide eluates from nine samples were
two or more grades stronger than the acid eluates (Table 2).
Formamide and acid eluates from 21 of the 22 samples with
reactive DATs were also tested against the same five panel
available to test one of the 22 samples. Formamide and acid
eluates from two of the samples with positive tube DATs
were non-reactive in the tube assay with all five panel cells.
Both formamide and acid eluates from 19 of the 22 samples
were reactive with panel cells in the tube assay. In 18 of the
19 samples the tube agglutination reaction grades of the
formamide and acid eluates were the same or differed by one
grade. An acid eluate from one of the 19 samples reacted 3+
with B cells and 2+ with screening cell 3. This was different
from that observed with the formamide eluate from the
same sample which reacted 1+ with B cells and weakly with
screening cell 3.
Eluting antibodies from umbilical cord RBCs
Eluates were prepared from umbilical cord blood samples
with formamide and acid methods. Almost all of the results
obtained for formamide and acid eluates were identical when
the eluates were tested against the cell panel. There was no
significant difference in strength or specificity of antibody
in eluates when evaluated in either the gel-microcolumn or
tube-agglutination assays. However, one sample eluted by
both the formamide and acid methods produced eluates that
were reactive and non-reactive, respectively, when tested
with the gel assay (sample 8, Table 3). The commercial kit
and formamide eluates of the other eight cord blood samples
contained either anti-A or anti-B, or a combination of both,
as determined using gel and tube assay methods.
The gel agglutination reaction grades of the formamide
and acid eluates were the same or differed by only one
grade in seven of the nine samples. The gel reactions of the
formamide eluates from two of the samples were two or more
grades stronger that reactions of the acid eluates (Table 3).
Reactivity patterns of eight of nine eluates prepared by either
acid or formamide were the same or within one grade of each

© 2002 Blackwell Science Ltd. Vox Sanguinis (2002) 83, 63–69

 Formamide elution of antibodies 67

Table 3 Acid and formamide eluates prepared from two of nine umbilical cord blood samples differed when tested in the gel-agglutination assay

Sample Gel DAT Cell 1 Cell 2 Cell 3 A1 Cell B Cell

number result acid/formamide acid/formamide acid/formamide acid/formamide acid/formamide

6 1+ 0/2+ 0/1+ 0/3+ 1+/1+ 0/1+

8 2+ 0/1+ 0/1+ 0/2+ 0/1+ 0/1+

Diagnosis was haemolytic disease of the newborn (HDN) for all samples.
Values represent the reaction grade of testing.
DAT, direct antiglobulin test.

Table 4 Results of testing eluates prepared from red blood cells (RBCs) sensitized with commercial polyclonal antisera using commercial acid kits and
formamide methodology and gel-agglutination assay

Acid eluate results Formamide eluate results

Screening Cell 1 Screening Cell 2 Screening Cell 3 Screening Cell 1 Screening Cell 2 Screening Cell 3
Gel DAT (homozygous*) (heterozygous) (antigen negative) (homozygous*) (heterozygous) (antigen negative)
Antisera results Cell a, Cell b Cell a, Cell b Cell a, Cell b Cell a, Cell b Cell a, Cell b Cell a, Cell b

Anti-C 4+ 4+, 4+ 4+, 4+ 0, 0 3+, 3+ 2+, 2+ 0, 0

Anti-E 4+ 4+, 4+ 4+, 4+ 0, 0 3+, 3+ 2+, 2+ 0, 0
Anti-e 3+ 1+, 3+ 2+, 3+ 0, 0 1+, 2+ 1+, 1+ 0, 0
Anti-K 2+ 2+, 1+ 2+, 1+ 0, 0 1+, 1+ 2+, 1+ 0, 0
Anti-k 1+ 4+, 4+ 3+, 3+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-Fya 2+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-Fyb 3+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-Jka 2+ 1+, 1+ 1+, 1+ 0, 0 1+, 1+ 1+, 1+ 0, 0
Anti-Jkb 2+ 2+, 2+ 1+, 2+ 0, 0 2+, 2+ 1+, 2+ 0, 0
Anti-s 4+ 3+, 3+ 1+, 1+ 0, 0 1+, 1+ 1+, 1+ 0, 0

*Eluates were tested against two homozygous antigen-positive cells (Screening Cell 1, Cells a and b), two heterozygous (Screening Cell 2, Cells a and b)
antigen-positive cells, and two antigen-negative cells (Screening Cell 3, Cells a and b).
Values represent the reaction grade.
DAT, direct antiglobulin test.

other when tested by the tube agglutination assay. Eluates

prepared with acid and formamide from sample 8 were non-
reactive in the tube assay.
Eluates from in vitro-sensitized RBCs
The formamide method eluted all IgG (Tables 4 and 5) and
IgM (Table 6) antibodies. These included Rh, Kell, Duffy, Kidd,
and MNSs blood group antibodies. All antibodies recovered
by both formamide and acid methods had the same specifi-
city and similar strengths when tested in either the gel- or
tube-agglutination assays. When eluates were tested in the
tube-agglutination assay, acid eluates generally reacted
slightly more strongly than formamide eluates. However, when
eluates prepared from RBCs sensitized with IgM monoclonal
antibodies were tested, in some cases formamide eluates
reacted more strongly than acid eluates.
Discussion
Formamide was found to be effective in removing Abs from
RBCs. The other reagents used for elution preparation were
found to be too cumbersome, too time-consuming, degrading
to the isolated antibodies, or did not yield good results
when tested with a panel of Rh-positive cells. Formamide
was clearly the most promising reagent. Recovery of antibody
specificity was demonstrated using samples from patients
with various diagnoses and umbilical cord bloods. The com-
mercial acid and formamide methods also did equally well
when both were compared using specific commercial antisera
with in vitro-sensitized antigen-positive RBCs. This indicated
that formamide was able to recover antibodies from many
clinically significant blood groups.
Autoantibodies (most of which were panagglutinins) in
adults with positive DATs were isolated efficiently by both

© 2002 Blackwell Science Ltd. Vox Sanguinis (2002) 83, 63 –69

68 L. Caruccio et al.

Table 5 Results of testing eluates prepared from red blood cells (RBCs) sensitized with commercial polyclonal antisera using commercial acid kits and
formamide methodology and tube-agglutination assay

Acid eluate results Formamide eluate results

Tube DAT Screening Cell 1 Screening Cell 2 Screening Cell 3 Screening Cell 1 Screening Cell 2 Screening Cell 3
results (Homozygous*) (Heterozygous) (Antigen Neg) (Homozygous*) (Heterozygous) (Antigen Neg)
Antisera (anti-IgG) Cell a, Cell b Cell a, Cell b Cell a, Cell b Cell a, Cell b Cell a, Cell b Cell a, Cell b

Anti-C 2+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 1+, 1+ 0, 0

Anti-E 3+ 3+, 2+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-e 2+ 2+, 1+ 2+, 2+ W, W H, H H, H W, W
Anti-K 1+ 3+, 3+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-k 2+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-Fya 1+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-Fyb 2+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 2+, 2+ 0, 0
Anti-Jka 1+ 2+, 2+ 2+, 2+ 0, 0 2+, 2+ 1+, 1+ 0, 0
Anti-Jkb 2+ 2+, 2+ 2+, 2+ 0, 0 1+, W 1+, W 0, 0
Anti-s 2+ 1+, W 2+, 2+ W, W 2+, 1+ 2+, W W, W

H = haemolysis; W = weak positive.

*Eluates were tested against two homozygous antigen-positive cells (Screening Cell 1, Cells a and b), two heterozygous (Screening Cell 2, Cells a and b)
antigen-positive cells, and two antigen-negative cells (Screening Cell 3, Cells a and b).
Values represent the reaction grade.
DAT, direct antiglobulin test.

Table 6 Tube agglutination results of testing eluates made from commercial the RBCs had to be washed with saline as many as six to
immunoglobulin M (IgM) monoclonal antisera-sensitized red blood cells eight times to remove all traces of jelly, and this extensive
(RBCs) in vitro washing may have dislodged weakly bound Abs. These
results may also have been a result of the higher level of
Acid Formamide
haemoglobin in the cord cells or the presence of fetal hae-
Antisera Agglutination eluates eluates
moglobin, as a reduction in antibody reaction grades was
noted with both elution methods. Despite the weaker reac-
Anti-C Positive 2+ 2+
Anti-E Positive W 2+ tions of cord blood eluates, anti-A and anti-B were detectable
Anti-c Positive W W with both methods.
Anti-e Positive W W IgG and IgM class Abs could be recovered by both forma-
Anti-K Positive 1+ W mide and acid-elution methods. In addition, all clinically sig-
Anti-A Positive W W nificant blood group Abs tested were recovered with good
Anti-B Positive W W specificity. Although IgG antibodies are generally considered
Anti-Jkb Positive W W more clinically significant than IgM antibodies, most com-
mercial antisera used for RBC phenotyping are IgM and an
W = weak positive.
elution method that is able to recover these antibodies intact
Eluates were prepared with commercial acid kits and formamide
would be very valuable [12]. Therefore, formamide-based
methodology and tested with antigen-positive cells at both immediate spin
elution may have special applications in industry and com-
and room temperature (the same result was obtained for each).
All testing was performed using only tube technology. mercial laboratories.
Values represent the reaction grade. There are three main disadvantages of using formamide as
an eluting agent. First, the eluates have a reddish colour from
free haemoglobin. However, this colour did not interfere with
formamide and acid methods. Anti-A and -B eluted from the interpretation of results of antibody identification using
umbilical cord blood did not react as strongly as the panag- either tube- or gel-agglutination assays. Previously pub-
glutinins or specific antibodies eluted from adult-blood lished elution methods have resulted in haemoglobin-stained
RBCs. This was true for eluates prepared using both the for- eluates and these gave acceptable results for antibody iden-
mamide and acid methods. This could be the result of tification [13]. Second, the formamide method produced RBC
Wharton’s jelly interfering with the eluate preparation, as cellular debris that might interfere with the test results in

© 2002 Blackwell Science Ltd. Vox Sanguinis (2002) 83, 63–69


antibody identification, especially in gel assays. The debris
can be removed with 0·22-µm filters or extra centrifugation
steps if necessary; however, these additional steps add extra
time and manipulation of the sample. Various commercially
available cartridges or columns can be used to remove forma-
mide in addition to the cellular, membranous debris. These
have the advantage that they also can clarify and concentrate
the eluate in addition to removing formamide. For example,
the spin-filter tubes of Cole-Parmer (Cole-Parmer Instrument
Company, Vernon Hills, IL), the centrifugal filter tubes of
Eppendorf (Brinkman Instruments, Inc., Westbury, NY), or
the Slide-A-Lyser small-volume dialysis units of Pierce
(Rockford, IL) are all appropriate for this purpose. The third
disadvantage is that formamide is considered by regulatory
agencies to be toxic and must be handled and disposed of with
greater care than the commercial acid-based kit reagents.
Most clinical laboratories have established methods for the
disposal of hazardous waste. However, in contrast to acid-
based elution methods, formamide does not require the use
of sodium azide as a preservative and therefore may be less
hazardous than commercial kits that require this potentially
dangerous reagent to maintain a reasonable shelf-life and
avoid microbial contamination.
Formamide is an acceptable method for the elution of Abs
from RBCs. Overall, the formamide method is fast, simple,
and reliable and could be performed by most technologists
with a minimum of training and experience and without major
problems. The cost of the formamide method is equivalent to
that of the commercial kits currently in use.
It should be possible to modify the formamide technique
described here to improve it and make it available as a com-
mercial kit. Any difficulties encountered using formamide are
easily overcome with simple modifications to the procedure.
The formamide method compares favourably with established
elution methods and may be recognized as an alternative
method in the future.
© 2002 Blackwell Science Ltd. Vox Sanguinis (2002) 83, 63 – 69
Formamide elution of antibodies 69
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