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HLA Antibody Specification Using Single-Antigen Beads—A Technical Solution for the Prozone Effect

Schnaidt, Martina1,4; Weinstock, Christof2; Jurisic, Marija1; Schmid-Horch, Barbara1; Ender, Andrea3; Wernet, Dorothee1

doi: 10.1097/TP.0b013e31822872dd
Rapid Communications

Background. Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect.

Methods. Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone.

Results. The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q.

Conclusion. Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

1 Center for Clinical Transfusion Medicine, University Hospital of Tübingen, Tübingen, Germany.

2 Department of Immunohaematology, German Red Cross Blood Service Rheinland-Pfalz und Saarland, Bad Kreuznach, Germany. Currently: Department of Transfusion Medicine, University Hospital Ulm; Institute for Clinical Transfusion Medicine and Immunogenetics Ulm; German Red Cross Blood Service Baden-Württemberg-Hessen, Institute Ulm, Germany.

3 Central Institute for Transfusion Medicine and Blood Donation, Katharinenhospital, Hospital of the City of Stuttgart, Stuttgart, Germany.

The authors declare no funding or conflicts of interest.

M.S. and C.W. contributed equally to this work and share the first authorship.

4 Address correspondence to: Martina Schnaidt, Ph.D., Center for Clinical Transfusion Medicine, University Hospital of Tübingen, Otfried-Müller-Str. 4/1, 72076 Tübingen, Germany.


M.S. and C.W. participated in research design, writing of the manuscript, and data analysis; M.J. participated in performance of the research; B.S.-H. and D.W. pariticipated in data analysis and writing of the manuscript; and A.E. participated in performance of the research and writing of the manuscript.

Received 6 October 2010. Revision requested 8 November 2010.

Accepted 6 June 2011.

Detection of human leukocyte antigen (HLA) antibodies and, particularly, precise determination of antibody specificity is required for patients awaiting kidney transplantation. In recent years, the introduction of solid-phase assays improved sensitivity in antibody detection compared with cell-based techniques. Substantial progress in correct specification, especially in patients with multiple HLA antibodies, was achieved using single HLA-antigen beads combined with the Luminex-technology (1). In this technique, however, high-titer antibodies were found to exhibit the high-dose prozone effect (prozone phenomenon), which is also known as the hook effect (2–4). This effect, if not recognized, can have serious consequences, because, especially, the strongest antibodies of patients are not, or only poorly, detected. To overcome this adverse effect, sera have to be retested after dilution (5), a cumbersome and cost increasing procedure.

Recently, Kosmoliaptsis et al. (4,6) reported that IgM antibodies may be responsible for the prozone effect. They hypothesized that competitive binding of IgM antibodies to the beads may block binding of IgG antibodies. Thus, they recommended the pretreatment of the sera with dithiothreitol (DTT) to destroy IgM antibodies.

When we tested serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we saw a prozone in serum but not in plasma. Because IgM antibodies are not damaged by EDTA, we assumed that DTT pretreatment exerts a different effect in addition to inactivation of IgM antibodies. A main property of EDTA is the ability to chelate metal ions. Of serum components, the complement system, in particular its component 1 (C1), is sensitive to both withdrawal of Ca2+ ions and cleavage by DTT. C1 binds to the Fc portion of IgG1 and IgG3 molecules. We hypothesized that (1) binding of C1 to IgG may block the binding of anti-IgG detection antibodies and (2) dissociation or destruction of C1 enables binding of the detection antibody. In this study, we tested this hypothesis.

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Effect of EDTA on Antibody Detection With SAB

Serum and EDTA plasma from the same venipuncture of a highly HLA-sensitized patient were tested in parallel. Surprisingly, some of the beads reaching the highest mean fluorescence intensities (MFIs) with EDTA plasma gave only weak positive or even negative results in serum.

Suspecting a prozone phenomenon, we tested this and another suspicious serum after 1:10 dilution, thereby confirming a prozone effect for some of the HLA antibody specificities (Fig. 1). The prozone was not detectable, when EDTA plasma from the same venipuncture was tested (Fig. 1). This led us to test the sera of four additional patients with a previously detected prozone effect as serum and with EDTA added (“EDTA serum”). The results show that addition of EDTA to serum also overcomes the prozone effect (Fig. 2). In our hands, most specificities showing a clear prozone effect had MFI values approximately more than 12,000 in EDTA serum. Specificities with MFI values less than 10,000 showed no or only little increase in their MFI after addition of EDTA (Fig. 2), indicating that addition of EDTA may improve the reactivity of high-titer HLA antibodies while leaving the reactivity of HLA antibodies with lower titers untouched.





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Influence of Different Serum Treatments on the Prozone Effect in SAB Testing

If our hypothesis regarding complement C1 is correct, not only EDTA but also other conditions adversely acting on C1 stability should eliminate the prozone. Accordingly, we tested sera containing IgG antibodies with a prozone effect after pretreatment with DTT, C1 inhibitor (C1INH), and after heat inactivation. Native serum and serum with EDTA added served as controls. Both, addition of DTT and heat inactivation abolished the prozone effect. C1INH was somewhat less effective but clearly abolished the prozone effect too (Fig. 3).



The results show that both destruction of the C1 molecule (heat, DTT) and cleavage of the C1r-C1s subunit from C1q (EDTA, C1INH) are effective in abolishing the prozone effect. In accordance with our hypothesis, the addition of EDTA and heat inactivation enhanced the reactivity of the IgM antibodies (data not shown).

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Reconstitution With Complement or C1q

To further test our hypothesis that the C1 molecule prevents the binding of the secondary antibodies and causes the prozone effect, we tested two sera, which had lost their prozone effect by heat inactivation, after adding freshly drawn serum as a source of C1. The reconstitution of these heat-inactivated sera restored the prozone effect (Fig. 3), showing that the prozone effect observed in our patients depended on a heat labile factor, which is present in normal native serum. In contrast to fresh serum, addition of purified C1q did not restore the prozone effect (Fig. 3), suggesting that only C1 as a whole but not stripped C1q can cause the prozone.

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Binding of C1q in SAB Testing

Native serum and serum after pretreatment with EDTA or DTT were tested in the single-antigen bead (SAB) with anti-C1q as secondary antibody. Native serum did not allow the detection of C1q, neither on beads with a prozone effect nor on beads without a prozone effect (Fig. 4). In the tests with EDTA serum, nearly all beads with a prozone effect were covered with C1q. Beads without a prozone had bound markedly less or no C1q. As expected, no C1q was detectable in tests with serum after DTT pretreatment, because DTT adversely affects C1q. The data indicate that the complete C1 molecule, as present in native serum, is not detected by anti-C1q secondary antibody. In EDTA serum, in contrast, where only the stripped C1q molecule is present, C1q was readily detected.



HLA-specific IgM antibodies bind C1q in direct proportion to their density on the bead, whereas HLA-specific IgG antibodies started to bind C1q only when a certain density of IgG on the bead surface was exceeded (Fig. 5).



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Solid-phase assays facilitate detection of and improve sensitivity for various analytes but may be impaired by a prozone effect observed in the presence of high analyte concentration. Thus, high concentrations of an analyte lead to false results, lower than expected or even negative. A widely used procedure to overcome the prozone caused by high concentrations of an analyte is retesting after dilution (5). The prozone effect also has been reported for antibody detection with antigen-coated beads in the Luminex technique (2–4). However, it is not understood how a high titer of specific antibody causes the prozone in bead-based systems, because the capture antigen (i.e., HLA antigen) is already fixed to the solid phase (i.e., bead) when the serum containing the analyte (i.e., HLA antibodies) is added. The number of HLA-specific antibodies binding to the beads should increase proportionally to the amount of antibody present in the serum, reaching a plateau as soon as all HLA antigens on the bead are covered with antibodies. Closely spaced HLA-specific antibodies on the bead surface, in theory, could cause steric hindrance for the anti-IgG detection antibody, which can be overcome by dilution of the HLA antibodies in the serum. However, steric hindrance by IgG cannot explain how addition of EDTA overcomes the prozone effect. An additional factor may cause the prozone phenomenon, a factor that is sensitive to EDTA and that is able to block the access for the secondary antibodies to the HLA antibodies bound to the beads.

Therefore, we put forward the hypothesis that the complement system component C1 could be the cause of the prozone phenomenon in SAB testing as follows: C1 binds to two or more closely adjacent IgG molecules and blocks the Fc portion of the HLA-specific antibodies for the approaching anti-IgG detection antibody. Two adjacent IgG molecules, bound in a proper distance, are needed for C1 to bind to IgG. When serum contains HLA antibodies with a lower titer, not all of the HLA antigens on the SAB are covered by IgG, leaving spaces between the IgG molecules, which prevent C1 from binding, but allow the anti-IgG detection antibodies to catch their targets. In contrast, when serum contains HLA-specific antibodies in abundance, all HLA antigens on the beads may be covered with IgG. This situation allows C1 to bind to most or even to all the antibodies on the bead and, thereby, competitively prevents binding of the anti-IgG detection antibodies. A similar mechanism is conceivable for IgM antibodies as suggested by preliminary data from our tests with HLA-specific IgM antibodies.

C1 consists of one C1q subcomponent, Ca2+ ions, and two subcomponents C1r and C1s each. C1q was described as “bouquet of tulips,” consisting of six globular heads with stalk-like protein chains ending in a central fibril-like stem (7). Two subcomponents C1r and two subcomponents C1s are positioned between the stalks of C1q, forming a Ca2+-dependent tetramer. The heads of C1q bind closely to the hinge region of the immunoglobulin molecule (8), and it is possible that the C1r-C1s tetramer blocks that part of the Fc region, which the anti-IgG detection antibody binds to. Withdrawal of Ca2+ dissociates C1r and C1s, leaving a stripped C1q subcomponent, which still is able to bind between immunoglobulins but does no longer block the access of secondary antibodies.

Several serum treatments known to dissociate or to destroy C1 successfully abolished the prozone effect: EDTA chelates Ca2+ ions, dissociating the C1r-C1s subunit from C1q. C1INH is a regulator of activated C1 present in normal human plasma. C1INH binds to activated C1s and dissociates the C1r-C1s-tetramer from the C1q molecule (7), thereby exerting a similar effect on C1 as Ca2+ withdrawal. Both, EDTA or C1INH added to serum abolished the prozone effect. DTT or heat inactivation destroys several serum proteins including C1 (9). Both addition of DTT and heat inactivation of serum abolished the prozone effect. DTT, EDTA, and protein denaturation by heat are unspecific treatments, which also act on serum proteins other than C1. C1INH not only acts on C1 but also inactivates the coagulation factors FXIIa and FXIa, tissue plasminogen activator, and kallikrein (10). However, to the best of our knowledge, C1 is the only serum protein that is sensitive to all four treatments: it is inactivated by 56°C, has disulfide bonds making it sensitive to DTT, depends on the presence of Ca2+ ions, and is a substrate of the serine-protease-inhibitor C1INH. Further support for a possible role of C1 in the prozone effect was provided by experiments in which the prozone effect was abolished by heat inactivation but was easily restored by the addition of fresh serum from nonimmunized male donors. Experiments in the past (11) showed that sera lost their complement activity in the antiglobulin test after storage at room temperature for 72 hr. We also noticed that a serum lost its prozone effect after storage at room temperature for 3 days (data not shown). Storage at +4°C or −22°C markedly slowed the deterioration of the complement, and storage at −55°C retained all the complement activity. Further, repeated freezing and thawing of samples is known to have detrimental effects not only on the stability of complement but also on the stability of a few IgG and some IgM antibodies. Thus, the conditions of transport and storage (duration and temperature) may determine whether a prozone effect is present or no longer detectable, when a serum is finally tested in the laboratory.

To detect complement on the SAB, a phycoerythrin-labeled anti-C1q antibody was used. Only HLA-specific antibodies with a prozone effect bound marked amounts of C1q. However, C1q was only detectable when EDTA serum was tested. Tests with native serum showed negative results. The reason for this apparent discrepancy again may lie in the structure of the C1 molecule. As mentioned earlier, in native serum, C1q forms a complex with C1r and C1s. In EDTA serum, the withdrawal of Ca2+ dissociates the C1 complex, leaving a stripped C1q molecule. So far, the epitopes of C1q to which anti-C1q binds to are not yet defined. We propose that the C1r-C1s tetramer may not only block the anti-IgG detection antibody but also block the anti-C1q antibody, preventing the detection of C1q. Thus, in native serum, C1 may be bound by HLA-specific antibodies with a prozone effect but cannot be detected by the anti-C1q used in this study. Further studies are needed to elucidate whether different anti-C1q antisera are able to detect native C1 on SAB.

HLA-specific IgG antibodies in EDTA serum showed different C1q-binding characteristics compared with IgM antibodies. IgM antibodies bound C1q proportional to their density on the beads (i.e., proportional to their MFI). In contrast, only IgG antibodies showing the prozone effect with high MFI bound C1q to the beads. These observations fit to our hypothesis that a high density of IgG on the bead surface is required to bind C1, and they further support our hypothesis that C1 causes the prozone effect in high-titer antibody sera.

Recently, data were published (4,6,12) stating that HLA-specific IgM antibodies may be responsible for the prozone effect, because addition of DTT (which destroys IgM molecules) abolished the effect. IgM antibodies competing with IgG for the HLA binding sites on the beads are a possible mechanism for the prozone effect. However, in the sera presented in this study, not only DTT but also addition of EDTA abolished the prozone effect. The structure of IgM antibodies does not depend on the presence of Ca2+, and EDTA does not destroy IgM (13). Furthermore, nearly all IgG and most IgM molecules are resistant to heat inactivation for 30 min at 56°C (14), but heat inactivation abolished the prozone effect. After heat inactivation, the prozone effect was restored by the addition of serum containing no HLA-specific IgM antibodies. Therefore, we are convinced that in the sera presented in this study, IgM antibodies did not play a role in the prozone effect.

In conclusion, the results of this study confirmed our hypothesis of the role of C1 in the prozone effect in SAB testing. First, the prozone effect was caused by a heat labile factor and was restored by the addition of normal serum containing no HLA-specific antibodies. Second, four different serum treatments abolished the prozone effect, all of them capable to dissociate or to destroy the complement component C1. Third, only high-titer IgG antibodies with a prozone effect were found to bind marked amounts of C1q.

In our opinion, the C1r-C1s tetramer of the native C1 molecule inhibits the binding of the anti-IgG detection antibody leading to the prozone effect. This view was supported by experiments, in which C1q was found on SAB but did not block the access of either anti-IgG or anti-C1q detection antibodies. Further, in contrast to the addition of fresh serum, addition of purified C1q to heat-inactivated serum did not restore the prozone effect but still allowed the detection of all specificities with a prozone effect.

Anyway, whether or not C1 alone or IgM, or both, cause the prozone effect, our data show that the use of EDTA plasma or addition of EDTA to serum is highly efficient in overcoming the prozone effect. This is in contrast to the manufacturers' recommendations to use blood collected without anticoagulant, stating that plasma may lead to lower MFI values as described by Norris et al. (15). When we compared the reactivity of antibodies not showing any prozone effect, sera with EDTA added gave comparable results to the native sera. Most notably, we never saw false-negative results because of addition of EDTA.

The prozone effect of high-titer HLA antibodies forces laboratories to test sera undiluted and diluted. The results of this study suggest that testing EDTA plasma instead of serum might avoid double work load and double costs. As long as the complement-dependent cytotoxicity (CDC) is performed routinely in pretransplant diagnostics, it is, however, not possible to change from serum to plasma samples. Requesting an additional EDTA sample may also not be suitable. Addition of EDTA to the patients' sera before SAB testing could be a practicable alternative.

Two benefits for laboratories are obvious: first, addition of EDTA may give more reliable results, and second, it will save costs, because retesting using diluted serum is not necessary. We hope that our report, based on few patient samples only, will encourage other diagnostic laboratories to test this easy approach, so that a larger number of samples tested in this way will lead to a general use of EDTA addition in HLA antibody specification by SAB testing.

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Serum samples were obtained from patients awaiting kidney transplantation at the University Hospital of Tübingen. According to the guidelines of Eurotransplant, sera of these patients are regularly tested with the CDC test for the presence of HLA antibodies. Sera showing a high percentage of panel reactive antibodies in CDC were selected for testing with SABs for further specification of antibodies directed to HLA class I and class II antigens. In addition, sera of highly HLA-immunized patients awaiting stem-cell transplantation were included. If results raised suspicion of a prozone effect, sera were retested at 1:10 dilution. In this study, a prozone effect was defined as a 2-fold increase of the MFI after 1:10 dilution (or other serum treatment). These investigations were initiated to optimize the antibody differentiation by SAB. Therefore, according to the ethics committee of the Medical Faculty, no ethical conflicts are concerned.

SAB testing was performed with Lifecodes Luminex single antigen class I and class II kits (Gen-Probe Transplant Diagnostics, Stamford, CT) or LABScreen HLA class I SAB (One Lambda Inc., Canoga/Los Angeles, CA). LABScreen Singles (One Lambda Inc.) were used for testing different serum treatments of sera containing only few HLA antibody specificities showing the prozone effect. The tests were performed according to the manufacturers' instructions. Evaluation was carried out with the software provided. The results were expressed as adjusted MFI. Tests for IgM antibodies were performed using LifeMatch anti-IgM secondary antibody (Gen-Probe Transplant Diagnostics). Testing for C1q was done with LABScreen SAB and C1qScreen (both from One Lambda Inc.), containing purified C1q and phycoerythrin-conjugated anti-C1q.

The following serum treatments were used to investigate the prozone effect in SAB testing:

  • 1:10 dilution in NaCl (0.9%)
  • Addition of EDTA: 5 μL EDTA solution (6%)+95 μL serum
  • Addition of an equal volume of 0.01 M DTT, 30 min at 37°C
  • Heat inactivation: 30 min at 56°C
  • Addition of C1INH: 10 μL C1INH (50 U/mL)+40 μL serum
  • Addition of an equal volume of freshly drawn AB serum from male, nonimmunized blood donors as a source of complement
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The authors thank Professor Gertrud Maria Hänsch, Institute for Immunology, University of Heidelberg, for the valuable information on complement. C1NH (Berinert) was generously provided by Dr. Rosenthal from CSL Behring. The C1q kit was a generous gift from One Lambda in cooperation with BmT, Meerbusch-Osterath. Finally, we are indebted to Hans-Georg Rammensee for critically reviewing the manuscript.

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Single-antigen bead assay; HLA antibodies; Prozone effect; Complement

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