ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC ...
ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC DIALYSIS
Andrea A. Zachary1, Donna P. Lucas1, Barbara Detrick 2, Mary S. Leffell1
Departments of Medicine1 and Pathology2
The Johns Hopkins University School of Medicine, Baltimore, MD
Andrea A. Zachary, PhD, D(ABHI)
The Johns Hopkins University School of Medicine
2041 E. Monument Street
Baltimore, MD 21205
phone: 410-614-8978 fax: 410-955-0431
Abbreviated title: Effect of IgM Elimination on Antibody Assays
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ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC DIALYSIS
SPI solid phase immunoassays
MFI mean fluorescence intensity
PBS phosphate buffered saline
Non-HLA-specific antibodies, IgM antibodies, and immune complexes can result in increased
background reactivity and can interfere with binding of HLA-specific antibodies, abrogating
clinical utility of the antibody test results. We present data here showing that immunoglobulins
of the IgM class can block binding of IgG HLA-specific antibodies and that precipitation and
removal of IgM by hypotonic dialysis can restore normal reactivity to some sera that have high
background in solid phase assays or unexplainable positive reactions in crossmatch tests. The
IgM-depleted fractions showed normal reactivity of negative controls and clear specificity and
reaction strengths that correlated with those of crossmatch tests. We also show that removal
of IgM by filtration reduces non-specific background but also reduces the strength of specific
reactions compared to those of IgM-depleted fractions obtained by hypotonic dialysis.
Hypotonic dialysis of sera with normal background levels and patterns of reactivity, does not
alter the specificity but may slightly reduce the strength of some specific reactions.
Two types of assays, differentiated by target, are used for the detection and identification of
HLA-specific antibodies:  the cytotoxicity and flow cytometric assays that use lymphocyte
targets and  solid phase immunoassays (SPI) that use soluble HLA molecule targets.
Lymphocyte-based assays are fraught with multiple technical issues including inappropriate
reactions due to reduced cell viability, variability in antigen expression, and the presence of
lymphocyte binding antibodies that are not specific for HLA. False positive reactions have been
particularly problematic when B lymphocytes are used as targets (1,2). SPI have resolved
many of the problems associated with lymphocyte-based assays, have superior sensitivity and
specificity, require small serum volumes, and can be partially or fully automated (3-7). The
result is the ability to obtain results very rapidly, detect the presence of antibody to donor HLA
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before clinical signs of antibody-mediated rejection appear, and more readily identify epitope-
specific antibodies. The high degree of sensitivity and specificity and the ease with which
antibody specificity can be determined permits differentiation of clinically relevant and irrelevant
crossmatches enhancing the clinical interpretation of test results.
Although SPI have fewer technical problems than do cell-based assays, like any immunologic
assay, they are not completely problem free (7-9). Binding of antibodies not-specific for HLA
antigens or of immune complexes may result in high background reactivity and may interfere
with binding of HLA-specific antibodies. The effects may be increased values for negative
controls, decreased values for positive controls, and/or alteration of the HLA specificities
detected. In turn, this may result in a failure to detect antibodies present and confound
interpretation of crossmatch results.
We have found that high background in SPI often occurs when there are high levels of IgM
autoantibodies or immune complexes present. Commercially available products designed to
reduce background resolve the issue in a some cases, but not most cases. Others have shown
that neither reducing agents nor heat aggregation eliminate IgM immunoglobulins completely
(10). IgM immunoglobulins are water insoluble euglobulins while IgG immunoglobulins are
water soluble pseudoglobulins. Dialyzing serum against distilled water results in precipitation of
IgM permitting an effective elimination of IgM (11). We present here data showing that
removal of IgM by hypotonic dialysis eliminates or reduces non-specific reactivity from SPI and
crossmatch tests sufficiently to provide clinically meaningful results.
Materials and Methods
Sera were from 16 renal renal transplant candidates. Selection of sera was based on the
following criteria: negative controls values above expected (4 sera), specificity and/or strength
of donor-specific antibody that did not correlate with crossmatch results (3 sera), or both (7
sera). Two sera that did not meet these criteria were also tested as controls. All sera were
tested as untreated sera and as the IgM-depleted pseudoglobulin fraction by multianalyte bead
assay on the Luminex® platform using panels appropriate for determining antibody specificity
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and strength. The total number of such tests exceeds the number of sera as some sera were
tested with both phenotype and single antigen panels and some sera were tested for both class
I and class II HLA specificity. Some of IgM-depleted fractions were also tested in crossmatch
tests against donor cells previously tested with untreated sera. A subset of sera had
immunoglobulin fractionation performed using spin columns and the IgG portion of each was
tested on the Luminex platform.
Sera were tested using phenotype panels (Lifematch class I, class II,, Tepnel LifeCodes,
Stamford, CT) and/or single antigen panels (Single antigen beads, One Lambda, Canoga Park,
CA) on the Luminex platform, according to the manufacturers’ instructions. Results were
expressed as specificity and as mean fluorescence intensity (MFI) values. A subset of sera were
tested with the DynaChip® ELISA system ( Invitrogen, Corp., Carlsbad, CA) according to
manufacturer’s instructions. Specificity analysis was performed manually by two to three highly
experienced individuals. Reaction strength in single antigen panel tests were made using raw
values to avoid the impact of elevated negative control values of the untreated sera.
Cytotoxicity tests were performed with positively selected T and B lymphocytes using
antiglobulin-enhanced and one wash procedures, respectively, as described previously (1).
Three color flow cytometric crossmatches were performed on a Becton-Dickinson FACSCalibur
as described, previously (5). The second antibody used was an IgG heavy chain-specific,
mouse monoclonal antibody (BD Biosciences, San Jose, CA). Results were expressed as a ratio
of the median channel fluorescence of the test serum to that of the negative control.
Dialysis of serum was performed using Pierce Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher
Scientific, Rockford, IL), according to manufacturer’s instructions. Briefly, hydrated cassettes
were loaded with 500w of the serum of interest and dialyzed against 4L of deionized water,
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overnight (14-16 hours) in the cold (2-8oC), with stirring. After overnight dialysis and when a
euglobulin precipitate was visible, the contents of the cassette was removed and centrifuged for
one hour at 15-20K x g at 2-8oC to collect the precipitated IgM-containing euglobulin fraction.
The precipitated euglobulin fraction was resolubilized in isotonic, buffered saline. The
supernatant fluid was removed and placed into a new dialysis cassette and back dialyzed
against 2 changes of 4L of phosphate buffered saline overnight at room temperature to restore
isotonicity. At the end of the second dialysis, the IgM-depleted fraction was removed from the
cassette and reserved for antibody testing.
Spin Column Immunoglobulin Fractionation
Separation of IgG and IgM immunoglobulin was performed using the 300K Nanosep®
Centrifugal Device (Pall Corporation, East Hills, NY). Briefly, 50Cl of serum was loaded into the
sample reservoir, covered, and spun at 14,000xg in a fixed angle centrifuge for 12 minutes. The
IgG portion of the serum was recovered from the lower chamber of the device.
Measurement of IgM Depletion
IgM immunoglobulin levels were measured in the serum and IgM-depleted fraction by rate
nephelometry (IMMAGE 800 System, Beckman Coulter, Inc, Fullerton, CA, USA) (12). These
tests showed that while there was some IgM remaining in the pseudoglobulin fraction, the
amount had been diminished by 67-82%.
Unless otherwise specified, IgM-depleted fraction refers to the soluble fraction remaining after
Comparison of Control Bead Results
Both the phenotype and single antigen panels have a positive control bead. The phenotype
panel has three negative control beads and the single antigen panel has one. Comparison of
the results obtained with these control beads for untreated sera and the IgM-depleted fractions
are given in Table 1. Some of the IgM-depleted fractions yielded different specificities than did
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the untreated sera. Therefore, the results in Table 1 have been separated into two categories
according to whether or not dialysis treatment resulted in a change in antibody specificity. For
some sera, changes were observed for only one of the two classes of specificity and the control
bead results were categorized, accordingly. In all categories, the mean values of the positive
controls were higher for the IgM-depleted fractions than for the untreated sera while the mean
values of the negative controls were lower. This was true not only for mean values but for
values of each individual serum.
The specificity assignments obtained with phenotype and single antigen panels, for cases where
the specificities of the untreated sera and IgM-depleted fractions differed, are given in tables 2
and 3, respectively. The specificity data for sera with no change in specificity after IgM
depletion by dialysis are not shown. In tests with phenotype panels, four sera, 5, 6, 8, and 16,
had no specificity identifiable in the untreated serum but had clearly identifiable specificities in
the IgM-depleted fraction. For serum 12, class I specificity could be determined only in the
IgM-depleted fraction. Class II specificity was determined in both the untreated and IgM-
depleted fractions but the specificities were substantially different (DR1 and DR4 in the
untreated serum and DR1 and DR52 in the IgM-depleted fraction). When a specificity was
determined, the MFI value given in Table 2 is for the highest ranking phenotype with reactivity
due to the assigned specificity. When specificity could not be assigned, the MFI is for the
highest ranking phenotype overall. In all cases where specificity changes occurred, the
reactivity strength was greater with IgM-depleted fraction than with untreated serum. There
were several sources of data indicating that the dialysis procedure did not produce an
artifactual change in specificity or reaction strength. Two control sera which did not have
increased background reactivity in the negative controls nor disparities in the strength of
antibody determined by antibody screening and crossmatch tests, showed no specificity
differences between the IgM-depleted fractions and untreated sera tested with phenotype
panels. As noted above, some sera demonstrated high background in tests for only one of two
HLA antibody specificity classes. There were two sera, 5 and 8, which had high background in
tests for class I-specific but not in tests for class II-specific antibodies. For both sera, IgM
depletion resulted in specificity changes only for class I-specific antibodies - i.e., those tests in
which high background had occurred with untreated sera. Further, specificity changes did not
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always occur after IgM-depletion of a serum with increased background. There was one serum
(serum 10) that had increased reactivity in the negative controls but had no specificity
differences between the IgM-depleted fraction and the untreated serum (data not shown). In
all cases except one, in which there was no specificity change, the reaction strength was less
for IgM-depleted fraction than for untreated serum. In the one exception, only one of two
specificities had lower strength in the IgM-depleted fraction than in the untreated serum. This
is in contrast to the results given above when the specificities obtained with the two
components were different and the reactions were uniformly stronger with the IgM-depleted
Ten untreated sera and corresponding IgM-depleted fractions were tested with single antigen
panels and all showed differences in the specificities of the five highest ranking beads (ranked
in order of descending strength). Seven of these pairs of components, sera 5-9, 13, and 16,
had complete differences - i.e., there were no beads in common among the top five - while for
3 pairs, 2, 3, and 11, there were some beads in common. In all but one case (serum 7) the
reactions were stronger with the IgM-depleted fractions than with the untreated sera, even for
beads that were the highest ranking with both fractions. In some cases the specificity
differences were not significant. For example, for serum 5, four of the five strongest reacting
beads of the untreated serum were not among the 5 highest ranking beads of the IgM-depleted
fraction but had strong reactions with the IgM-depleted fraction with MFI values ranging from
11,767 to 16,899. However, the A3002 bead was among the five highest ranking beads for the
untreated serum 5 (MFI = 13271) but was one of the lower ranking beads for the IgM-depleted
fraction (MFI= 5278). Importantly, the rank order of the beads was changed in all cases.
Correlation With Crossmatch Results
We have established correlations between MFI values and crossmatch strength based on
numerous comparisons. Our investigation was undertaken, in part, because of cases in which
the strength of reactions in the antibody identification tests could not be reconciled with those
of the crossmatch tests. There were 10 sera tested in solid phase assays and used in 12
crossmatch tests. The IgM-depleted fractions of nine sera were tested in solid phase assays
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and seven of the crossmatches were repeated using IgM-depleted fractions. Table 4 provides
the MFI values for donor-specific antibodies and the strength of crossmatches with the
respective donors. Reactivity of the untreated sera included:  MFIs that were too low to
account for the strength of ten crossmatch tests - nine CDC (sera 3, 5, 6 in 2 crossmatches, 7,
8, 9, 11, and 16) and one flow cytometric (serum 12) crossmatch;  a positive flow cytometric
crossmatch with T cells in the absence of class I-specific antibody (serum 7); and  positive
allogeneic and autologous crossmatch test results (serum 14). Using the IgM-depleted
fractions, antibody identification tests were repeated for nine sera and seven crossmatch tests
were repeated. The MFI values for the IgM-depleted fractions were increased for 8 sera (sera
3, 5, 6, 8, 9, 12, and 16) to become proportional to the strength of the crossmatches performed
with the untreated sera. The IgM-depleted fraction showed no change in titer in three repeated
crossmatch tests involving sera 5, 11, and 12, and a slight decrease in the titer of serum 16, all
of which were consistent with the MFIs obtained with the IgM-depleted fractions of those sera.
The IgM-depleted fraction of serum 7 yielded a negative cytotoxicity crossmatch and a flow
cytometric crossmatch that was negative with T cells and of reduced strength, compared to that
obtained with untreated serum, with B cells. The crossmatch results for the IgM-depleted
fraction of serum 7 were now consistent with both the untreated serum and the IgM-depleted
fraction of serum 7. Both allogeneic and autologous crossmatches performed with untreated
serum 14 were positive while those performed with the IgM-depleted fraction were negative
and correlated with the low MFI values obtained in the solid phase assays of the untreated
serum. For this serum, the solid phase immunoassays were not repeated with the IgM-
depleted fraction since the results of control beads with the untreated serum were within
normal ranges. In all cases, the IgM depleted fractions permitted reconciliation of the results of
crossmatch and solid phase tests and meaningful clinical interpretation.
Tests of Sequential Sera
We compared the results of tests of the untreated serum and IgM-depleted fraction of serum
12 with the results of tests of two previous sera from the same serum donor. These data are
given in Table 5. The MFIs for the class I phenotype panel control beads and for the B locus
antigens for the six strongest reacting phenotypes are provided. The patient had demonstrated
antibody to HLA-B7 for several months up to the day before transplantation (T-1). Two days
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after transplantation (T+2) we observed an increase in the reactivity of the negative control
beads of the class I phenotype panel and an even greater increase in the reactivity of the
negative control beads of the cII phenotype panel (data not shown). Four days after
transplantation (T+4), the increased background reactivity continued and no specificity could be
determined for class I reactivity. The HLA-B7-bearing phenotypes were now found among the
15 lowest reacting phenotypes. The predominant class II-specific antibody, which had been
DR17 was now DR1 and DR4 (data not shown). A test of the IgM-depleted fraction of the T+4
serum showed a restoration of normal reactivity in the negative controls and of the B7, DR17
specificity patterns seen in previous sera. It further revealed a very substantial increase in the
strength of those antibodies.
In an attempt to shorten the procedure time, we evaluated spin columns that provide
separation of IgG and IgM fractions and also tested the impact of reducing the times of the two
incubations of the hypotonic dialysis procedure. Results obtained with the IgM-depleted
fraction and IgG fraction obtained by spin column separation of nine sera are shown in Table 6.
Spin column separation was as effective as hypotonic dialysis in reducing background and
providing appropriate levels of reactivity with control beads in the solid phase assays. However,
in all cases, the HLA-specific reactivity of the IgM-depleted fraction obtained by dialysis was
stronger than that of the IgG fraction obtained by spin column separation. Also, there were 3
sera (3, 6, and 16) for which the 5 strongest reacting single antigens beads were not the same
for the two different preparations.
We have found that precipitation of the euglobulin fraction requires the overnight (ca. 16 hour)
dialysis in the cold. To assess the time of back dialysis against PBS needed to restore
isotonicity, we tested aliquots of the pseudoglobulin fraction taken immediately after dialysis
against distilled water and taken at two hour intervals after the start of dialysis against PBS.
Washed, packed red cells were resuspended in the various fractions and examined for lysis.
We found that after 4 hours of dialysis against PBS, no red cells lysis occurred. Further,
lymphocytotoxicity tests using the preparation obtained after 4 hours of back dialysis against
PBS yielded no inappropriate lymphocyte death.
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Tests of Sera in ELISA
DynaChip™ is an automated ELISA performed on glass microchips that contain single class I
and class II phenotypes. We tested 5 untreated sera on the DynaChip™ platform. For all 5
sera, the background reactivity was significantly reduced compared to tests of the untreated
sera on the Luminex platform. Four of the five sera were panreactive making it difficult to
determine antibody specificity precisely. However, the fifth serum was the untreated T+4
serum shown in Table 6. Tests of this serum on the DynaChip™ showed almost no reactivity in
the negative control and a clear pattern of specificity with B7 being the predominant antibody.
That is, the DynaChip™ results of tests of the untreated serum were very similar to the results
of Luminex tests of the IgM-depleted fraction of that serum.
Cell-based crossmatch tests have been used for more than 40 years to determine the strength
of donor-reactive antibody. Increasingly, it is being demonstrated that solid phase
immunoassays can be used to assess both the specificity and strength of antibodies to HLA and,
thus, provide an interpretive guide for cell-based crossmatches and, in many cases, a virtual
crossmatch (7, 13-16). We have shown here that solid phase assays can be confounded by the
presence of blocking factors comprised of IgM immunoglobulin and/or soluble immune
complexes. This interference may appear as inappropriately high reactivity with negative
control beads, as reactivity that lacks a definable pattern of HLA specificity, and/or as reactivity
that does not correlate in strength with that of crossmatch tests. We present a simple method
utilizing hypotonic dialysis that effectively depletes IgM blocking factors which, in turn, provides
improved specificity determination and more accurate correlation with donor crossmatches.
Dialysis of serum against distilled water precipitates the euglobulin fraction of serum and has
been used historically as a means of reducing or eliminating IgM (11). We have demonstrated
here that IgM can produce substantial test interference making it difficult or impossible to
determine antibody specificity and strength. We have further shown that the IgM-depleted
fraction obtained by hypotonic dialysis has a substantially reduced background level of
reactivity and has antibody specificity and strength that correlate with crossmatch results.
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Several lines of evidence indicate that the results obtained with the IgM-depleted fraction were
correct. First, in the multianalyte bead assays, IgM-depleted fractions had higher values for
the positive control beads and lower values for negative control beads than did the euglobulin
fractions. These values were more in line with expected values for valid assays. Second, the
IgM-depleted fractions yielded antibody identification test results that correlated with those of
crossmatch tests in both antibody specificity and strength. In most cases reported here, the
reactivity of the IgM-depleted fraction was different from that of untreated serum in the solid
phase assays while crossmatch reactivity was the same for both fractions. However, in two
cases, the opposite occurred. That is, the reactivity of the crossmatch test was different for the
IgM-depleted fraction. Third, tests of sequential sera from an individual patient showed that
the IgM-depleted fraction of a serum with high background reactivity yielded results that were
in line with those of other sera that were from the same patient but that did not have high
The IgM-depleted fraction from sera with high background reactivity had HLA specificity that
differed from that of the untreated serum. However, no change in specificity occurred following
dialysis of sera with normal reactivity with control beads. The effect on specificity was variable.
For sera with high background in tests of either class I-specific antibodies or class II-specific
antibodies but not both, changes in specificity occurred only with the tests that had high
background with untreated serum. There was also variability in the degree of impact on
specificity. For some sera, the same beads were positive with both untreated serum and the
IgM-depleted fraction but the ordering of the beads, when listed in descending order of
strength, was changed and often clustering of specificities was evident only with the IgM-
depleted fraction. For some sera, the ordering of the beads was affected more drastically. For
example, with serum 2, the three strongest reacting beads for the IgM-depleted fraction were
alleles of HLA-A2, with MFI values of 21-23,000. These three beads were also positive with the
untreated serum but were ranked 13th, 26th, and 36th, in bead order with MFI values of 4-8,000.
The most drastic differences occurred with serum 6 where the five highest ranking beads in
tests of the IgM-depleted fraction, all with MFI values ca. 23,000, were negative with the
untreated serum with raw MFI values of 312-585. In all cases, the predominant antibodies
gave stronger reactions with the IgM-depleted fraction than with untreated serum. This was
not true for all antibodies. Some “apparent” antibodies that gave moderate reactions with
untreated serum, had weaker reactions with the IgM-depleted fraction.
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The hypotonic dialysis procedure is simple but takes two days. For the small volumes of serum
treated here, we found that isotonicity sufficient for tests of cells, was restored in four hours of
dialysis against phosphate buffered saline and that no such back dialysis was necessary for solid
phase assays. However, we found that a clear euglobulin precipitate was evident only after a
minimum of 18-20 hours of dialysis against distilled water. Separation of immunoglobulins
using spin columns with a filter permeable to IgG but not to IgM requires less than two hours.
The results of the IgG fractions obtained with these columns yielded specificities comparable to
the IgM-depleted fractions obtained by hypotonic dialysis but with appreciably lower reactivity
strength. This may have been due to IgM overloading the filter and blocking permeation of
some of the IgG. This suggests that the spin column procedure would be helpful in clarifying
antibody specificity but not with determining antibody strength. Traditional methods to reduce
IgM reactivity involve aggregation through heating or the reduction of disulfide bonds by agents
such as dithiothreitol and dithioerythritol. Heat-inactivation of serum is not suitable for solid
phase testing since the aggregates formed may bind non-specifically while IgM reduction
involves the use of carcinogenic agents and may cause the loss of some IgG. Further, neither
technique will eliminate IgM reactivity completely (10). As has been described by others, a
small amount of IgM was left in the IgM-depleted portion of the dialyzed serum. However, the
IgM-depleted portion of the serum was free from the background activity of the untreated
serum and provided clear specificity determination.
Several questions remain to be answered. It appears that some component in the euglobulin
fraction is binding to the Luminex beads and blocking the binding of HLA-specific antibody. The
euglobulin portion contains other serum components in addition to IgM and the relative
contribution of those components, if any, is unknown. Another issue is why the non-specific
binding may occur with assays for class I only, class II only, or both. This does not appear to be
due to binding of an HLA-specific IgM antibody since this should not result in reactivity with
negative control beads. Additionally, we have tested the euglobulin fraction of two sera and did
not find HLA-specific antibody. Reduced reactivity seen in the DynaChip™ assay which is
performed on a glass microchip, suggests that nonspecific reactivity in the Luminex assay may
be the result of binding of a serum component to the polystyrene of the beads. However, one
would expect that binding would occur equally with beads bearing class I and those bearing
class II antigens. Further, treatment of these sera with commercially available reagents to
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abrogate non-specific binding to beads did not reduce background nor restore normal reactivity
to the sera. The results obtained here indicate that there are differences in the negative control
beads in the class I and class II kits for both the phenotype and single antigen panels.
In summary, we have demonstrated that hypotonic dialysis is an effective way to reduce or
eliminate non-specific binding to Luminex beads and permit specific binding of antibodies to the
HLA antigens. A more rapid method for achieving this is via filter separation however, this
method appears to reduce the amount of HLA-specific antibody present. Utilizing a matrix of
glass rather than polystyrene may also be an option for reducing non-specific binding to plastic
but this approach needs further investigation.
page 13 of 22
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4th ed. Mt. Laurel, NJ: The American Society for Histocompatibility and
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Human Immunology. Boca Raton, FL: CRC Press; 1997, p 478-519.
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Immunology. 2008. In press.
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Table 1. Control Value Means for Untreated Sera and IgM-Depleted Fractions
Group Sera with No Specificity Change1 Sera with Changes
Ab Class Panel Type Control2 No. Untreated No. Untreated
PC 131954 18027 13856
I Phenotype 4
NC 4 81-129 55-90 322-866
PC 14745 18740 14254
II Phenotype 6 3
NC 117-324 71-245 367-1139
PC 10780 13827 10702
I Single Antigen NC 2 371 94 4 420
PC:NC 31 190 33
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PC 11173 12421 9792
II Single Antigen NC 4 186 124 3 147
PC:NC 147 107 152
Refers to whether or not there was a difference in the antibody specificities identified in the
untreated serum and IgM-depleted fractions.
PC: positive control, NC: negative control. For the phenotype panels, the means of the range
of the three negative controls is provided.
Water soluble fraction obtained after hypotonic dialysis
Values are means of the mean fluorescence intensity (MFI) values
No. refers to the number of pairs of untreated sera and IgM-depleted fractions.
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Table 2. Sera With Changed Specificity - Phenotype Panels
Untreated Sera IgM-Depleted Fraction1
Specificity Maximum MFI2 Specificity Maximum MFI2
5 no pattern 17473 B7 CREG 23710
6 no pattern 20758 B7 CREG 25774
panreactive - DR9, DR10
7 19397, 17930 panreactive - DR7, DR9 strongest 22873, 20256
8 no pattern 15582 A3, A11, B7 24342, 24690, 24787
9 DR1, 10, 53 6008, 9808, 15057 DR10, 53 22284, 17301
12 no pattern for cI, DR1, 4 17129, 11355, 11898 B7 CREG, DR1, DR52 20464, 24752, 24172
16 no pattern 15754 DR1, 2, 9 22250, 21501, 21501
Water soluble fraction obtained after hypotonic dialysis
Values are for the highest reaction due to antibody of the listed specificity or, when not specificity was determined, for the highest
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MFI values are the raw values; to conserve space, all DRB1 alleles are given as DRB and no asterisks are included for any allele. Differences between
the untreated serum and the IgM-Depleted Fraction are shown in bold.
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Table 4. Crossmatch and Antibody Identification Data
Untreated Sera IgM-Depleted Fraction2
Pt Mismatches Pan 1
MFIs CDCXM3 FCXM4 MFIs CDCXM3 FCXM4
3 DQA5 S 1878 B: 256 19898
5 B35 S 5729 T>512 17301 T>512
6 B37, B49 S 8612, 9585 T: 32 12065, 12883
A29, A68, B27, 9688, 1495, 9730, 10258, 20374,
6 S T: 128
Cw2 4849 21096, 6079
7 DR11, DR15, DQ7 S 938, 1199, 9853 B: 2 T4, B7 459, 461, 9032 NEG T- B:4
4798, 13254, 19000, 13721,
8 A30, B58, Cw10 S T:128 NT
9 DR4, DR53, DQ7 S 1354, 5862, 584 B: 512 722, 14084, 17155
9 DR1, DQ5 S 7716, 8770 NEG 4693, 6197
11 DR1, DR11, DQ5 S 568, 4114, 5121 B: 8 114, 1200, 15265 B: 8
12 B7, DR17 P 9986, 4036 T:25, B:90 20464, 22930
allo: T64, B88,
14 A2, B7, B13 P 533, 1887, 533 auto
auto: T73, B82
16 DR1, DQ5 S 6442, 1873 B>512 17303, 21226 B: 256
Panel from which MFI values were determined, P: phenotype, S: single antigen. Values are given as maximum MFI for the
Water soluble fraction obtained after hypotonic dialysis
Titers for cytotoxicity crossmatches
Values are ratios of median channel of test serum to that of the negative control
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