Prozone Effect Research Articles

Evaluation of a species-specific C-reactive protein assay for the dog on the ABX Pentra 400 clinical chemistry analyzer

BackgroundA canine-specific immunoturbidimetric CRP assay, Gentian Canine CRP Immunoassay) with species-specific controls and calibrators was introduced and recently evaluated on the clinical chemistry analyzer Abbott Architect c4000 as well as on the Olympus AU600.Aims of our study were 1) to independently evaluate the canine-specific CRP assay on the ABX Pentra 400 clinical chemistry analyzer in comparison to the previously validated human-based immunoturbidimetric assay (Randox Canine CRP assay) and 2) to assess the impact of different sample types (serum versus heparinized plasma) on the results.Imprecision, accuracy, interference and the prozone effect were determined using samples from healthy and diseased dogs (n = 278). The Randox Canine CRP assay calibrated with canine specific control calibration material served as a reference method. Additionally, the impact of the sample type (serum and lithium heparin) was evaluated based on samples of healthy and diseased dogs (n = 49) in a second part of the study.ResultsLinearity was present for CRP concentrations ranging from 4 to 281 mg/l. For clinically relevant CRP concentrations of 7–281 mg/l, recovery ranged between 90 and 105% and intra- and inter-assay CVs ranged between 0.68% - 12.12% and 0.88% - 7.84%, respectively. CV was thus lower than 12.16%, i.e. the desired CV% based on biological variation. Interference was not present up to a concentration of 5 g/l hemoglobin, 800 mg/l bilirubin and 10 g/l triglycerides. No prozone effect occurred up to 676 mg/l CRP. Method comparison study revealed a Spearman’s rank correlation coefficient of rs = 0.98 and a mean constant bias of 5.2%. The sample type had a significant (P = 0.008) but clinically not relevant impact on the results (median CRP of 30.9 mg/l in lithium heparin plasma versus 31.4 mg/l in serum).ConclusionsThe species-specific Gentian Canine CRP Immunoassay reliably detects canine CRP on the ABX Pentra 400 clinical chemistry analyzer whereby both serum and heparin plasma can be used. The quality criteria reached on the Abbott Architect c4000 and Olympus AU600 could be met.

Unexpected Positive Prospective Crossmatches in Organ Transplant.

Preformed donor-specific antibodies against human leukocyte antigen can induce antibody-mediated rejection after organ transplant. Hence, future transplant recipients undergo pretransplant screening for preformed antibodies (ie, virtual crossmatch). Subsequently, prospective (analytic) crossmatching is performed using conventional, complement-dependent cytotoxicity assays and/or flow cytometry-based methods. The present article reviews factors that must be considered when unexpected, positive, prospective crossmatches are observed. First, the prozone effect caused by the interference of complement or immunoglobulin M must be abrogated by treating the serum with moderate heat, dilution, hypotonic dialysis, EDTA, or dithiothreitol. Second, the physician must check for the presence of potentially interfering autoantibodies (in a context of autoimmune disease or human immunodeficiency virus infection) or therapeutic antibodies (such as rituximab and antithymocyte globulin). In conclusion, knowledge of each assay's technical characteristics will enable the physician to reliably interpret any discrepancies. The reasons for an unexpected, positive, prospective crossmatch must be elucidated before transplant to ensure efficient organ allocation and optimize patient outcomes.

Desensitization strategies in the patient awaiting heart transplantation.

The aim of this review is to discuss the impact of antibody sensitization on heart transplant recipients. We will discuss the current state of antibody identification, antibody strength and potential antibody pathogenicity. The prevalence of antibody sensitization has increased with increased utilization of mechanical circulatory support (MCS). Data on MCS-related antibody sensitization are reviewed. Important considerations to minimize the prozone effect in antibody testing will be discussed. We present an overview of strategies for desensitization in solid organ transplantation. Current protocols for desensitization therapies will be presented. Finally, future research avenues, including potential further refinement of the detection of human leukocyte antigen (HLA) epitopes to improve donor-recipient immunologic matching, future direction for prevention of donor-specific antibodies (DSAs), the potential role of non-HLA antibodies and further potential refinement of the immune system for management of sensitized patient will be proposed. Antibody sensitization is a key immunologic issue prior to and after heart transplantation. Therapies aimed at immunomodulation, termed desensitization strategies, may improve immunologic matching at the time of heart transplantation.

Improvement in the definition of anti-HLA antibody profile in highly sensitized patients.

The definition of anti-HLA antibody profile in highly sensitized patients on a waiting list is crucial when virtual crossmatch is used in organ allocation systems, but also when used to identify the true deleterious anti-HLA antibodies. Here we propose different levels of risk based on the results of anti-HLA antibody testing in neat serum (N) and after sera dilution (DIL) and C1q test in 18 highly sensitized patients. This group was heterogeneous in terms of anti-HLA antibody titers and their ability to fix complement. After dilution, 15 out of 18 patients (83.3%) showed a reduction of positive bead counts whereas 4 patients showed a prozone effect and complement fixation was demonstrated. The high dilution of sera and ascertaining the complement fixation allow the accurate definition of risk anti-HLA antibody profiles in highly sensitized patients, as demonstrated in 5 of the sensitized patients who were transplanted.

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

The prozone effect exerted by the complement-binding anti-Lea on anti-D.

BACKGROUND:Prozone phenomenon is seen with very high-titer antibodies in an immune serum.AIM:The prozone effect on anti-D by a low-titer anti-Lea was investigated associated with neonatal jaundice.MATERIALS AND METHODS:Standard methods were used in investigations.RESULTS:The child was born at full-term developed mild jaundice. With weak direct antiglobulin test+, her indirect serum bilirubin was progressed to 27.5 mg/dL in 48 h. Anti-D and anti-Lea were detected in the mother. Both these antibodies were detected in the child's serum though the eluate from red blood cells (RBCs) contained only anti-D. Mother's anti-D was masked by anti-Lea if the RBCs possessed both the antigens together. Anti-D was revealed only with D-positive RBCs lacking Lea or if the serum was modified by mixing with Lea+ saliva or was heated at 56°C or fortified with citrate solution.CONCLUSION:An anti-D showed prozone effect exerted by the complement-fixing anti-Lea in the test.

Development of an Imperacer (Immuno-PCR) assay combining broad assay range and excellent sensitivity to support development of an immuno modulator antibody drug

Event Abstract Back to Event Development of an Imperacer (Immuno-PCR) assay combining broad assay range and excellent sensitivity to support development of an immuno modulator antibody drug Shuangping Shi1, Mark Spengler2*, Beena Punnamoottil2, Michael Adler3 and Don (Dong-Hun) Lee1 1 Merck Research Labs, Bioanalytical Development, Biologics, United States 2 Chimera Biotec, Project Management, Germany 3 Chimera Biotec, Assay Development, Germany Purpose: Biologics such as therapeutic antibody drugs are often administered in wide concentration ranges for dose-response evaluation. Safety and potency considerations demand low dosing in clinical trials. In contrast, higher dosing is applied in preclinical safety assessment for fast cleared drugs. Thus, sensitive pharmacokinetic analysis with broad quantification range is required. Method: An Imperacer assay for quantification of a therapeutic antibody for cancer treatment was developed. The assay applies tailored antibody-DNA conjugates for target detection and subsequent exponential DNA amplification for signal generation. Results: A quantification range of 2 – 32,768 pg/ml target antibody was demonstrated with accuracy and precision <25%. Selectivity at spiked 6 - 10 pg/ml was challenged with 10 healthy human serum samples. Dilution linearity confirmed that concentrations >5 μg/ml can be diluted into the assay range with acceptable recovery despite Prozone (hook) effect starting at approx. 1 μg/ml. Conclusions: Exponential signal amplification provides a combination of excellent sensitivity and broad assay range for the Imperacer platform. As only one assay is required for >5 orders of magnitude target concentration, the need for multiple quantification windows in different technologies is circumvented. This significantly reduces bioanalytical sample testing time and effort during all phases in the development of Biologics. Assay optimization is still in progress and will be adapted towards further study requirements prior to method validation. Keywords: pharmacokinetic, pk, Clinical support, immuno-PCR, IPCR, ultra sensitive immunoassays Conference: EUFEMED 2017, London, United Kingdom, 17 May - 19 May, 2017. Presentation Type: Poster Topic: EUFEMED 2017 CONFERENCE Citation: Shi S, Spengler M, Punnamoottil B, Adler M and Lee D (2019). Development of an Imperacer (Immuno-PCR) assay combining broad assay range and excellent sensitivity to support development of an immuno modulator antibody drug. Front. Pharmacol. Conference Abstract: EUFEMED 2017. doi: 10.3389/conf.fphar.2017.62.00007 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Aug 2017; Published Online: 25 Jan 2019. * Correspondence: Dr. Mark Spengler, Chimera Biotec, Project Management, Bremen, Germany, spengler@chimera-biotec.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shuangping Shi Mark Spengler Beena Punnamoottil Michael Adler Don (Dong-Hun) Lee Google Shuangping Shi Mark Spengler Beena Punnamoottil Michael Adler Don (Dong-Hun) Lee Google Scholar Shuangping Shi Mark Spengler Beena Punnamoottil Michael Adler Don (Dong-Hun) Lee PubMed Shuangping Shi Mark Spengler Beena Punnamoottil Michael Adler Don (Dong-Hun) Lee Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

P028 Uncovering the invisible; pre-treatment of sera with EDTA relieves prozone effect

Aim High titer HLA antibodies in patient sera can be falsely assessed due to interfering factors, also known as the prozone phenomenon, causing the masking of such antibodies. In this study we assessed the elimination of prozone by treatment of sera with DTT, EDTA and dilution. Methods Sera from 10 patients were treated with DTT, 6% EDTA, and 1:10 dilution. Antibody screening was performed by Luminex single antigen bead (SAB) assay. Effect of each treatment on the negative and positive controls, false-positive, and false-negative beads were compared to one another as well as to the untreated serum. Results Patient sera treated with DTT, EDTA and diluted at 1:10, all showed elimination of interfering factors and resolved the prozone effect. Comparison of the three treatment approaches showed that, DTT resolved the prozone, however it appeared to cause a slight increase in the mean fluorescent intensity (MFI) of the Luminex negative control (NC) bead. EDTA treatment eliminates prozone effect and has no effect on the NC bead. The 1:10 dilution also can resolve prozone, however the sensitivity can be compromised especially for the lesser strength antibodies. Conclusion In circumstances where prozone phenomenon is suspected, such as in the pre-transplant evaluation of highly sensitized patients, or during post-transplant monitoring for detection of donor specific antibodies, DTT, EDTA, and dilution are all effective strategies in elimination of interfering factors. However in addition to a higher NC, DTT-treatment, unlike EDTA, requires an additional incubation step with the sera (45 min) which affects the overall turnaround time. Dilution of serum can affect the sensitivity of the assay and in addition, often one dilution might not be sufficient for an accurate assessment of prozone, and performing multiple dilutions can prove to be costly. Routine treatment of sera with EDTA can accurately assess antibody specificities without affecting the sensitivity, turn around time and test cost.

P127 Detection of MICA antibodies: Comparison between MICA Screening and Single Antigen tests

Aim The presence of MICA antibodies is associated with worse graft outcome in solid organ transplant. Here, we aim to compare two MICA antibody detection tests: MICA Screening vs. Single Antigen Beads Based Test (SAB). Methods 20 sera with or without EDTA treatment were tested for MICA antibodies by screening (Onelambda LABScreen Mixed) or SAB tests (LABScreen MICA). Ratio of 2 in the screening test and MFI 1,000 in SAB test were used as the cutoff, respectively. Results We determined if EDTA treatment impact MICA SAB testing. Out of 15 samples, 7 samples displayed 23 MICA antibodies in EDTA treated or non-treated sera. In general, no significant difference of MFI (median fluorescence intensity) for MICA antibodies was observed between EDTA treated and non-treated sera. We did observe MFI of one MICA antibody increased from 9167 to 20566 upon EDTA treatment, suggesting this antibody showing prozone effect. We also found EDTA treatment increased MFI of positive control bead significantly (mean 6675 vs 9180). Next we compared results of MICA SAB testing on 20 EDTA treated sera with screening testing. We failed to identify the association between SAB test and screening test results (fig 1A). 4 sera, out of 8 MICA screening+/SAB- sera did not display HLA antibodies. 3 of these 4 patients had no sensitization events, suggesting positivity in these 3 patients is likely due to high background in MICA screening test. The mean ratio of screening testing on sera which were positive in SAB testing trended higher than sera having negative SAB test results, but the difference didn’t reach significance (fig 1B). Strong MICA antibodies (MFI>10,000) detected by SAB testing in two sera showed ratio >100 in screening testing, suggest both these assays can detect strong antibodies. Conclusions EDTA treatment may remove prozone effect for strong antibodies in MICA SAB testing. The screening testing tends to have high background, but strong MICA antibodies can be detected by both screening and SAB testing. The MICA SAB test is more specific than MICA screening test. Download : Download high-res image (71KB) Download : Download full-size image

P137 The importance of re-evaluating the HLA antibody profile and auto-antibody status when the flow cytometry crossmatch result is not as expected

Aim The flow cytometry crossmatch (FCXM) is a decision-maker for organ transplantation. A positive FCXM against T-Cells and/or B-Cells increases the risk for rejection. For this reason, a “positive” FCXM often eliminates the patient from consideration for transplant. The listing of unacceptable HLA (UAgs) reduces the probability for an unexpected positive FCXM. The FCXM and processes to define UAgs are not perfect. Herein, we provide three cases in which the transplant decision changes with additional laboratory testing and the way the FCXM is reported. Methods A standard three-color FCXM uses PerCP-CD3 and PE-CD19 (BD Bioscience) to label T- and B-Cells, respectively, and FITC- Fab’2 goat anti-human IgG (Jackson Lab) to detect antibody binding. A median channel difference (MCD) of 35 for T-Cells and 65 for B-Cells is the threshold for positive. Single antibody beads (Thermo Fisher) were used for HLA antibody identification. UAgs are listed based on mean fluorescence intensity (MFI) of 1000 for Class I and 3000 for Class II. Results All the cases were evaluations for deceased-donor kidney transplantation. Case 1: A re-transplant candidate with 100% cPRA had an unexpected strong positive T-FCXM (MCD over 400) with no known Class I DSA. A serum tested at a 1/10 dilution showed two Class I DSAs, A2 and B57 with a combined peak MFI of 10,303. Case 2: A B-FCXM was reported as strong positive with a MCD of 378. The patient had strong autoantibody reacting with his B-Cells (MCD 270). Subsequent B-FCXMs were reported as uninterpretable because of a history of auto-antibody reacting with B-Cells with no known DSA. The patient was successfully transplanted. Case 3. A patient with a negative antibody history and no auto-antibody had an unexpected positive T-FCXM (MCD 192). The B-FCXM was Negative. A new HLA antibody profile was negative. A new auto-crossmatch was T-FCXM positive(MCD 73) and B-FCXM negative. Subsequent T-FCXMs were reported as uninterpretable because of a history of auto-antibody reacting with T-Cells with no known DSA. The patient was successfully transplanted. Conclusions 1. A prozone effect must be evaluated when listing UAgs. 2. Unexpected FCXM results should be investigated. 3. The clinical relevance of the FCXM result must be communicated to prevent a patient being removed from consideration, inappropriately.

P152 Prozone phenomena may skew the strength of Donor Specific Antibody (DSA)

Aim Positive Complement Dependent Cytotoxicity (CDC) and Flow Cytometry Crossmatch (FCXM) usually correlate with high titer of Donor Specific Antibodies (DSA). Now a days assignment of relevant DSA is based on solid phase assay (SPA), in which false low/negative results can be observed due to prozone effect. Here we present three cases underwent a workup for living related kidney transplant that their crossmatch data does not correlate with the single antigen (SA) Median Fluorescent Intensity (MFI) value. Method B cell and/or T cell AHG-CDC, FCXM and antibody identification by SPA were performed for three living related pairs. Results Two patients show positive T cell AHG-CDC crossmatch and one shows positive B cell CDC crossmatch with 8 reaction strength at 1:8 dilution and positive FCXM with very high Median Channel Shift (MCS). However the DSA result was negative for one patient and the other two patients showed A2 (891 MFI) and DQ6 (1299 MFI) respectively. The SA testing indicated possible skewed MFI value, therefore all serum were diluted 1:4 to overcome a prozone effect if present. Conclusion MFI value of the SPA was not correlated with the high MCS and positive CDC crossmatch in undiluted serum due prozone phenomena of high antibody concentration. Therefore serum dilution should be considered if strong crossmatch reactivity observed.

P032 Determining the minimum EDTA concentration necessary to eliminate prozone effect in the single antigen bead (SAB) assay

Aim A major limitation of the single antigen bead (SAB) assay is its susceptibility to interference by complement activation, which inhibits binding of anti-IgG-PE to HLA specific antibodies. This interference, known as prozone effect, can reduce MFI values leading to false negative reactions. Schnaidt et. al. (2011) described a method to eliminate prozone by treating sera with EDTA to chelate calcium, preventing complement activation. However, anecdotal evidence suggests that the method is not always effective. This may be due to differences in EDTA concentration used by different labs given the original description is ambiguous and lacks sufficient detail to follow the protocol. The goal of this study was to determine the minimum EDTA concentration required to eliminate prozone. Methods Six well characterized, prozone +ve sera (3 class I and 3 class II HLA) were selected. Prozone +ve specificities were defined as >5000 MFI increase in SAB assay reactivity upon titration. Sera were treated with several concentrations of dipotassium EDTA (1.2–6 mM) or PBS control and were tested in parallel using LABScreen SAB assay. MFI values of prozone +ve specificities were then compared. Results A total of 52 prozone +ve specificities (28 class I and 24 class II HLA) were identified in titration studies. Prozone was eliminated for all 52 specificities when sera were treated with either 6 mM or 3 mM EDTA (Fig. 1). In contrast, treatment with either 2 mM or 1.2 mM EDTA was ineffective (Fig. 1). Prozone −ve specificities were not affected by EDTA treatment at any concentrations used. Conclusion EDTA treatment of sera is effective at eliminating prozone if appropriate EDTA concentrations are used. Interestingly, our experiments demonstrate that the threshold at which EDTA is effective is very distinct (3 mM is effective while 2 mM is ineffective). Since EDTA forms a 1:1 chelate complex with calcium, the concentration threshold of EDTA required to eliminate prozone is consistent with physiologic serum calcium concentration (2.1–2.6 mM). Download : Download high-res image (369KB) Download : Download full-size image

P054 EDTA treatment of serum eliminates interference in flow cytometric crossmatches

Aim A recurrent problem in our flow crossmatch (FXM) is a skewing, or shift to the left, of the T cell population, indicating interference by some component of patient serum. The suspected interfering agents are high levels of complement proteins or IgM antibodies. Previously, we have tried to correct this problem using heat inactivation and spin column treatment of the serum, or running the FXM with nonpronase treated cells. However, each of these options had drawbacks and did not consistently provide accurate results. Using nonpronase cells reduces sensitivity and causes occasional false positive results. Ethylenediaminetetraacetic acid (EDTA) has been shown to eliminate interfering substances, specifically complement proteins, in patient serum that causes a prozone or blocking effect during antibody testing, especially those patients with high titer antibodies (Schnaidt M, et. al. Transplantation, 2011). We report the use of EDTA as a pretreatment of patient serum to remove interfering agents in the T cell FXM. Methods Three color FXM using the Halifax protocol were performed with pronase treated (15 min/37 °C) donor cells. For FXM showing skewed T cells, sera were pretreated with EDTA (95 μl serum + 5 μ l EDTA) and then added to cells as in standard FXM. Results Twelve patient serum samples showed significant T cell skewing in FXM against seven different donor cells, but this shift to the left was eradicated by EDTA treatment in all of these FXM. For example, in one FXM there was a reduction in gated CD3 + T cells from 80% in the negative control serum compared to 15% in the neat, untreated patient serum – but when this patient serum was treated with EDTA, the CD3 + T cell population was restored to 80%. Furthermore, in this particular FXM, there was a difference in FITC median fluorescence intensity (MFI) of 120 channels when comparing the neat to the EDTA treated patient serum. EDTA treatment did not affect negative and positive control sera as the CD3 + T cells and FITC MFI levels were unchanged in FXM comparing EDTA to untreated serum controls. Conclusion EDTA treatment of serum is an easy, quick, inexpensive and effective way to remove elevated levels of complement proteins, IgM and possibly other proteins from patient sera which interfere with T cell FXM. EDTA treatment provides accurate interpretation of the T cell FXM.

P008 Case scenario, performance differences of the two commercially available solid phase single antigen reagents

Aim Luminex single antigen technology for antibody identification has enabled laboratories to provide highly accurate virtual crossmatch results prior to transplantation. Both manufacturers, One Lambda (OL) and Immucor (IC) single antigen reagent panels differ in the HLA alleles composite and density. We report here two cases that single antigen panel for each behave significantly different. Method Serum of Patient (RH) and (AM) were tested with OL and IC single antigen panels. Flow cytometry and CDC crossmatchs of both patients were performed with their corresponding donors. Results Serum of (RH) and (AM) tested with OL single antigen panel that showed the following DSA’s, anti DQB1*06:03 (2510 MFI) and anti A2 (891 MFI), B41 (1631 MFI) respectively. CDC and flow crossmatch were highly positive for both sera despite low Median Fluorescent Intensity (MFI) of the identified DSA. The same sera were tested with IC single antigen panel; patients (RH) and (AM) showed anti DQB1*06:03 (17946 MFI) and anti A2 (10500 MFI) respectively. Furthermore, both sera were diluted 1:4 and re-tested with OL single antigen panel. The identified DSA strength in the diluted sera for both patients showed higher MFI values for A2 (3048 MFI), B41 (2863 MFI) for AM patient and DQB1*06:03 (10503 MFI) for RH. Conclusion Significantly, higher prozone effect was observed with OL single antigen panel than in IC. Serum to bead-volume ratio may have contributed to the lower correlation between OL versus IC. Testing patients sera by more than one manufacturer is an advantageous in determination of antibody strength and improves correlation with the crossmatch results.

A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect.Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer.

The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks.

Previous work has shown that Suc1/Cks proteins can promote the hyperphosphorylation of primed Cdk1 substrates through the formation of ternary Cdk1-Cks-phosphosubstrate complexes. This raises the possibility that Cks proteins might be able to both facilitate and interfere with hyperphosphorylation through a mechanism analogous to the prozone effect in antigen-antibody interactions, with substoichiometric Cks promoting the formation of Cdk1-Cks-phosphosubstrate complexes and suprastoichiometric Cks instead promoting the formation of Cdk1-Cks and Cks-phosphosubstrate complexes. We tested this hypothesis through a combination of theory, proof-of-principle experiments with oligonucleotide annealing, and experiments on the interaction of Xenopus cyclin B1-Cdk1-Cks2 with Wee1A in vitro and in Xenopus extracts. Our findings help explain why both Cks under-expression and overexpression interfere with cell-cycle progression and provide insight into the regulation of the Cdk1 system.

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information

The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen-antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen-antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.

Comparison of EDTA versus DTT treatment in overcoming the prozone effect in a Luminex-based HLA antibody assay

Aim One of the limitations of solid-phase HLA antibody assays is the false low/negative reading in a subset of samples with high titer/strength HLA antibodies, a phenomenon known as the prozone effect. The goal of this study was to compare the efficacy of ethylenediaminetetraacetic acid (EDTA) vs. dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect in solid-phase HLA antibody assays on the Luminex platform. Methods A total of 21 serum samples were tested by single-antigen bead assays (One Lambda), including 14 previously identified prozone samples (3 class I, 11 class II), 6 previously identified non-prozone samples with HLA antibodies (3 each for class I and II), and 1 negative sample. Serum was treated either by adding EDTA to a final concentration of 5 mM, or by incubating with 5 mM DTT for 30 min at 37 °C. To evaluate the efficacy of prozone effect reversal on HLA single antigen beads assay, fourfold serial dilutions, from neat to 1:256, were tested for each positive sample, using PBS as a diluent. Results Prozone effects were reduced in all 14 samples by either EDTA or DTT treatment. Results revealed sharp increases of MFI values in treated serum samples at neat, compared to non-treated samples. However, EDTA and DTT treatment were not equally efficient in overcoming the prozone effect. EDTA had nearly 100% reversal of prozone effect in all the tested samples, estimated by the ratio of MFI at neat to peak MFI at any given dilution. In contrast, the efficacy by DTT treatment ranged from 47% to 100%. The difference between the treatments was particularly notable in samples with strong prozone effects, defined by peak MFI values beyond a 1:16 dilution with untreated samples. Consequently, EDTA-treated serum samples showed virtually no further MFI increase when diluted, while some DTT-treated serum samples still had MFI increases when diluted, an indicator of residual prozone effect. For the negative control sample as well as non-prozone samples, comparable results were obtained from EDTA, DTT and non-treated serum. Conclusions Our study indicates that EDTA is more efficient in reversing the prozone effect, compared to DTT treatment. Furthermore, the stability of EDTA and the simplicity and robustness of serum treatment provide additional advantages over DTT for routine clinical testing.

Treating sera with ethylenediaminetetraacetic acid: A promising technical solution for the complement-mediated prozone effect in anti-hla antibody detection by single antigen bead assay

Aim The Luminex-single antigen bead (SAB) assay is considered the most sensitive method for the identification of anti-HLA antibodies (Ab). However, false negative results can occur due to the prozone effect (PrE) leading to underestimation of some high titer anti-HLA Ab. The aim of this study was to evaluate the ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement in eliminating the PrE in anti-HLA Ab identification by SAB. Method Ten patients with PRAs > 65% were included in the study. Neat and EDTA treated samples were tested using SAB for either HLA class I (n=14) or class II (n = 10) specificities. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in EDTA treated versus neat sera was considered relevant to PrE. The PrE was confirmed with 1:10 dilution and with dithiothreitol treatment of the sera. To confirm the clinical significance of the new identified Ab, sera with defined PrE and MFI value 1000 in neat sera were further tested with AHG-CDC crossmatch against donor cells expressing the relevant HLA. Results PrE was found in 7/10 patients concerning either HLA class I (n = 4) or class II (n = 3) Ab specificities. Using an MFI = 1000 as a cut off, six ‘new’ HLA specificities were identified either HLA class I (HLA-A∗01:01, A∗34:02, A∗11:02) or class II (DQB1∗03:01, DQB1∗03:02, DQB1∗03:03). However, a significant shift of MFI value of HLA Ab class I and II was observed. More precisely, 38 HLA-class I Ab directed against HLA-A (n = 31) and HLA-B (n = 7) showed a significant mean MFI value shift from 5162 (266 lower value) to 20207. Regarding HLA class II Ab, 26 directed against HLA-DQ (n = 23) and HLA-DR (n = 3) showed a significant shift from MFI 4419 (113 lower value) to 23795. New HLA Ab specificities detected after EDTA treatment were confirmed with a strong positive AHG-CDC crossmatch. Conclusion Taking into account that selection of potential recipients pre-Tx as well as the follow up post-Tx is based to HLA Ab identification, these preliminary results highlight the importance of using EDTA treatment of the patient’s sera prior to SAB assay, in order to abolish the prozone effect in HLA antibody detection.

The complement interference phenomenon as a cause of underestimation of high titer anti-HLA antibodies in a renal transplant recipient

Single antigen bead assay is the most sensitive laboratory tool for the anti-HLA antibody (Ab) identification in renal transplant recipients. However, this assay was shown to be prone to the prozone effect (PrE), giving false negative results in sera with high titer anti-HLA Ab. The ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement was described to eliminate the PrE allowing the anti-HLA Ab identification. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in neat versus EDTA treated sera was considered relevant to PrE. We report a case of chronic Ab mediated rejection in a renal transplant recipient with anti-HLA-DQA1∗05:05,DQB1∗03:01 donor specific Ab (DSA), revealed after treatment of the sera with EDTA. Female patient, 36 years old, diagnosed with juvenile nephronophthisis, received a first graft from a living related donor in 1991. During the first year post transplantation the patient experienced two biopsy proven reversible rejection episodes and until 2010 she had no other evidence of graft malfunction. De novo DSA were detected in 2010, as shown on the table. In 2015, due to inconsistency between the sudden elevation of serum creatinine levels and absence of DSA, the patient’s sera were further analyzed after EDTA treatment. DSA with high MFI were detected post-EDTA treatment. The presence of DSA was confirmed with positive B-Flow crossmatch with the donor. Also, a kidney biopsy on 03/26/2015 showed chronic antibody mediated rejection with C4d deposition. Taking into account that the follow up post-transplantation is based on HLA Ab detection and identification, these results highlight the importance of using patients’ sera with EDTA treatment to abolish the PrE before the performance of single antigen bead assay, in order to detect the presence of HLA antibodies. Download : Download full-size image