Polyethylene Glycol
A New Potentiator of Red Blood Cell Antigen-Antibody Reactions
SANDRA J. NANCE, M.S., MT(ASCP) S.B.B. AND GEORGE GARRATTY, PH.D., F.I.M.L.S., M.R.C.PATH.
A new technic using polyethylene glycol (PEG) was developed
to detect and enhance weak red blood cell/antibody reactions.
Twenty percent PEG of 4,000 molecular weight was found to be
optimal. Weakly reactive antibodies (n = 25) were tested by
PEG, Polybrene®, and low ionic strength saline (LISS); 64%
were strongest in PEG, 28% reacted equally in PEG as in Poly-
brene or LISS, 8% reacted weaker in PEG than in Polybrene or
LISS. Stronger antibodies (n = 11) were titrated and compared
in the three technics; in 10 of 11 titrations, PEG was better or
equal to Polybrene and LISS. The false positive rate with the
use of PEG and anti-IgG was 1.5% with the use of random sera.
Sera from patients (n = 24) with hemolytic transfusion reactions
and no detectable antibody by routine technics were tested; two
sera had specific antibodies by the PEG technic. This new technic
should be a valuable aid in the detection and identification of
weak antibodies. (Key words: Polyethylene glycol (PEG); Im-
munohematology; RBC antigen-antibody reactions; Agglutina-
tion; Potentiators) Am J Clin Pathol 1987; 87: 633-635
POLYETHYLENE GLYCOL, (PEG) is a water-soluble
polymer used extensively in industry as a lubricant for
rubber molds, textile fibers, and ceramics, and in many
other applications. PEG has been used for immunoglob-
ulin precipitation and immune complex detection.3 PEG
has also been used in the fractionation of plasma,10 in
antisera concentration,6 and in diagnostic test kits as a
precipitin accelerator. We investigated its use as a poten-
tiator of red blood cell (RBC) antigen-antibody reactions.8
Materials and Methods
Indirect PEG Procedure
Patients' or donors' sera (two drops) were mixed with
one drop of 4% reagent or donors' RBCs and four drops
of PEG solution. The optimal PEG solution was found
to be of 4,000 molecular weight (Sigma Chemical Com-
pany, St. Louis, MO) at a 20% suspension in phosphate-
buffered saline (PBS). The serum/PEG/RBC mixture was
incubated at 37 °C for 15 minutes, then washed four times
with PBS. It was important not to centrifuge after the
37 °C incubation because the presence of PEG made it
Received on May 16, 1986; received revised manuscript and accepted
for publication July 14, 1986.
Address reprint requests to Ms. Nance: Los Angeles-Orange Counties
Region, 1130 South Vermont Avenue, Los Angeles, California 90006.
633
American Red Cross Blood Services, Los Angeles-Orange
Counties Region, Los Angeles, California
difficult to resuspend the RBCs if they were centrifuged.
After washing, anti-IgG was added and the tubes centri-
fuged at 1,000X g for 20 seconds. Appropriate weak pos-
itive and negative controls were always tested in parallel.
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The weak positive control is a dilution of anti-D that is
nonreactive in low ionic strength saline (LISS) but is re-
active in the PEG and Polybrene® technics.
Direct PEG Procedure
One drop 4% patient RBCs was added to each of two
tubes. Two drops of 5% inert AB sera was added to one
tube and two drops of patient's serum to the other. Four
drops of PEG solution was added, mixed, and incubated
at 37 °C for 15 minutes. The RBCs were washed four
times with PBS; anti-IgG was added and the tubes cen-
trifuged at 1,000X g for 20 seconds.
Other Serologic Methods
The serologic methods used in this study have been
described previously.2,712 Titrations were scored by the
method of Petz and Garratty. 9
Enzyme-Linked Antiglobulin Test (ELAT)
This test was performed as described previously."
Results
Random sera from hospital patients (n = 104) and do-
nors (n = 100), with no demonstrable antibodies by a
LISS technic, were tested with PEG and Polybrene tech-
nics. We used anti-IgG throughout because initial tests
with the use of polyspecific antihuman globulin yielded
14% nonspecific reactions by the PEG technic that were
found to result from complement sensitization. All of the
donor sera were negative by the PEG technic, but two
were positive by Polybrene: of these, one antibody was
identified as anti-M, and the other's specificity could not
be determined. Four of the 104 random hospital patients'
 AJ.C.P.-May 1987
634 NANCE AND GARRATTY

Table 1. Comparison of LISS, Polybrene, and PEG in Tests with Weak Antibodies
Anti- LISS Polybrene PEG Anti- LISS Polybrene PEG

D m 1+ 1+ Le" Vl+ '/2 + 1+

C m 0 IV2+ Lea m 1+ 1+
E Vl+ m 1+ P, 2+ 1+ 2'/2+
E 1+ '/2 + 2+ Fy" 2+ 2+ 2V2+
E 1+ 1+ IV2+ Fy" IV2+ 1+ 2+
E '/2+ 1+ 1+ F/ IV2+ 1+ 1+
E '/2+ 1+ 1+ Fy" 2+ IV2+ 1+
c 1+ 1+ IV2+ Jka Vl+ m 1+
c V2+ 1+ 1+ Jka 0 0 '/2 +
c Vl+ 0 1+ Jk" m Vl + 1+
e 1+ 1+ 2+ Jkb l'/2 + 2+ 2'/2 +
M m 1V2+ IV2+ Hy Vl+ 0 '/2+
S Vl + 1V2+ 2'/2+

m = microscopically positive.

sera reacted by the PEG technic: 1 showed anti-Jkb spec- PEG, Polybrene, and LISS technics. Higher or equal titers

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ificity; the specificity of 2 sera could not be determined; were obtained with PEG in 10 of 11 (91%) antibody ti-
and 1 was insufficient for further study. With the use of trations (Table 2).
the Polybrene technic, seven patients' sera reacted, but Twenty-four cases of hemolytic transfusion reaction
either we were unable to identify the specificity of the with no antibody detected by routine procedures5 were
antibodies or there was not sufficient volume for further tested by less common procedures (4% ficin capillary,
studies. Polybrene) and our new PEG technic. In one case, we
Because we hoped that PEG could act as a potentiator were able to detect an anti-Jka in PEG and Polybrene. In
of weak reactions, we tested 25 weak antibodies that were another case, an anti-e was detected by 4%ficincapillary,
microscopically to 2+ reactive in a LISS technic at the Polybrene, and PEG. The other 22 cases were negative
antiglobulin phase of testing. The specificities included by all technics.
anti-D, -C, -E, -c, -e, -M, -S, -Fy3, -Jka, -Lea, -P,, and Often patients who were suspected of having Coombs-
-Hy. Parallel tests were performed in PEG, Polybrene, negative autoimmune hemolytic anemia, and whose
and LISS technics using antigen-positive and antigen- RBCs were sensitized with IgG that was only detectable
negative RBCs (Table 1). Sixteen (64%) reacted strongest by a direct ELAT,4 only two had a positive direct PEG
by the PEG procedure; seven (28%) reacted equally by test. Two of the ten patients also had a positive direct
PEG, Polybrene, or LISS; two (8%) reacted weaker by the Polybrene test.
PEG procedure than with Polybrene or LISS. None of
the antibodies tested were nonreactive by the PEG technic. Discussion
Eleven stronger antibodies were titrated, and master Several potentiating media have been described since
dilutions of each antibody were tested in parallel with albumin,1 a recent example being Polybrene.7 Our intitial
experiences with PEG suggests it may be more sensitive
Table 2. Comparison of LISS, Polybrene, and PEG in than Polybrene and yield fewer false positive reactions.
Tests with Strong Antibodies When an anti-IgG was used, the incidence of false pos-
itive reactions associated with the indirect PEG technic
Titration Score was 0% with donor sera and 2.9% with sera from hospital
Antisera LISS Polybrene PEG
patients, yielding a total false positive rate of 1.5%. This
compared favorably with the false positive rate of 2.5%
Anti-D,C 33 41 53 associated with Polybrene.
Anti-c 16 29 30 Sixty-four percent of weak antibodies were enhanced
Anti-E 33 45 67
Anti-K. 44 36 52 by PEG. Compared with Polybrene, significantly higher
Anti-S 23 25 35 titration scores (differences of 10 or greater) were seen in
Anti-Fy" 30 30 50 7 of 11 (64%) stronger antibodies when PEG was used,
Anti-Fyb 26 25 37
Anti-Fyb 1 20 9 and in 10 of the 11 (91%) PEG had an equal or higher
Anti-Jk" 14 9 18 score. In comparing LISS with PEG, 6 of 11 (55%) anti-
Anti-Jk" 15 11 21 bodies showed significantly higher scores with PEG.
Anti-Jkb 10 18 19
Again, 10 of 11 antibodies gave equal or higher titration
Mean score 22.3 26.3 35.5 scores with PEG as compared with LISS.
Vol. 87 • No. 5 BRIEF SCIENTIFIC REPORTS
The indirect PEG test was useful in detecting alloan-
tibodies that had caused hemolytic transfusion reactions
and were not detectable by routine procedures. We have
previously described Polybrene and the 4%ficincapillary
technics as also being useful in this area.5 We do not know
why such clinically significant antibodies are detectable
by these unusual technics but not routine procedures, in-
cluding the use of antiglobulin tests using enzyme-treated
RBCs.
The direct PEG test was not as sensitive as the
ELAT 4 " in detecting small amounts of RBC-bound IgG
autoantibody in tests with RBCs from ten patients sus-
pected of having Coombs-negative autoimmune hemo-
lytic anemia. All the RBCs tested had negative direct an-
tiglobulin tests but positive direct ELATs. Only two cases
had positive direct PEG tests. Unfortunately, we were un-
able to obtain further samples from these two patients
when they were in hematologic remission to determine if
the results of the direct PEG test had become negative.
In summary, the indirect PEG technic enhanced the
reactivities of most alloantibodies tested and detected two
clinically significant antibodies not detectable by routine
procedures. We do not suggest, at present, that this test
should replace any of the commonly used procedures, but
it may be useful as a supplementary test in the detection
of weak antibodies. The PEG technic may also be useful
in enhancing reactivity of weak, rare typing reagents, pro-
vided adequate quality control of test sera is performed.
Presence of HTLV-III Antibodies in Immune
Serum Globulin Preparations
MYLA LAI-GOLDMAN, M.D., JAMES H. McBRIDE, PH.D., PETER J . HOWANITZ, M.D.,
DENIS O. RODGERSON, PH.D., JAMIE A. MILES, AND JAMES B. PETER, M.D., PH.D.
The authors tested 15 immune serum globulin pharmaceutical
preparations for antibody reactivity to human T cell lympho-
trophic virus type HI (HTLV-III) by the Abbott® immunoen-
zymometric assay (IEMA). Although no evidence of HTLV-III
infectivity has appeared after injection of similar preparations
into humans, the authors found all samples IEMA reactive. Upon
dilution, the authors demonstrated parallel decreases of antibody
reactivity among two samples of gamma globulin, the Abbott-
positive control, and a markedly reactive patient specimen.
Received May 16, 1986; received revised manuscript and accepted for
publication August 6, 1986.
Address reprint requests to Dr. Howanitz: UCLA, A2-250 CHS, Los
Angeles, California 90024.
635
Acknowledgment. The authors acknowledge the assistance of Robert
Nance, Chief Executive Officer, Diamedex International, Santa Ana,
California, who provided the first PEG solution tested in this study.
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Department of Pathology, UCLA Medical Center and
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Am J Clin Pathol 1987; 87: 635-639