Prozone Effect Research Articles

Evaluation of the analytical performance of the novel NS-Prime system and examination of temperature stability of fecal transferrin compared with fecal hemoglobin as biomarkers in a colon cancer screening program

ObjectiveTo examine the analytical aspects of fecal transferrin (Tf) and hemoglobin (Hb) measured on the NS-Prime analyzer for use in a colon cancer screening program. Designs and methodsMethod evaluation and temperature stability studies for fecal Tf and Hb were completed. A method comparison was carried out against the NS-Plus system using samples collected from 254 screening program participants. A further 200 samples were analyzed to help determine suitable reference limits for fecal Tf using these systems. ResultsThe assay for fecal Tf showed acceptable linearity, precision, and recovery, and showed minimal carryover with low potential for impact by the prozone effect. The 95th percentile for fecal Tf obtained for the reference population was 4.9µg/g feces. The collection device sufficiently maintained fecal Tf and Hb stability for at least 7 days at room temperature, 4°C, and −20°C. Fecal Tf and Hb were most stable at 4°C and −20°C, but showed considerable loss (20–40%) of both proteins at 37°C within the first 7 days. Mixing small amounts of blood into diluted fecal samples maintained at 37°C for various time periods showed >50% loss of both proteins within 1h of incubation. ConclusionsThe NS-Prime analyzer showed acceptable performance for fecal Tf and Hb. These studies suggest that use of both Tf and Hb together as biomarkers will result in higher positivity rates, but this may not be attributed to greater stability of Tf over Hb in human feces.

Detection of alloantibody-mediated complement activation: A diagnostic advance in monitoring kidney transplant rejection?

ObjectiveAntibody-mediated rejection (ABMR) is an important cause of kidney allograft injury. In the last two decades, detection of complement split product C4d along transplant capillaries, a footprint of antibody-mediated classical complement activation, has evolved as a useful diagnostic marker of ABMR. While it was recognized that ABMR may occur also in the absence of C4d, numerous studies have shown that C4d deposition may indicate a more severe rejection phenotype associated with poor graft survival. Such studies suggest a possible diagnostic benefit of ex vivo monitoring the complement-activating capability of circulating alloantibodies. Design and methodsWe reviewed the literature between 1993 and 2015, focusing on in vivo (biopsy work-up) and in vitro detection (modified bead array technology) of HLA antibody-triggered classical complement activation in kidney transplantation. ResultsPrecise HLA antibody detection methods, in particular Luminex-based single antigen bead (SAB) assays, have provided a valuable basis for the design of techniques for in vitro detection of HLA antibody-triggered complement activation reflected by C1q, C4 or C3 split product deposition to the bead surface. Establishing such assays it was recognized that deposition of complement products to SAB, which critically depends on antibody binding strength, may be a cardinal trigger of the prozone effect, a troublesome in vitro artifact caused by a steric interference with IgG detection reagents. False-low IgG results, especially on SAB with extensive antibody binding, have to be considered when interpreting studies analyzing the diagnostic value of complement in relation to standard IgG detection. Levels of complement-fixing donor-specific antibodies (DSA) were shown to correlate with the results of standard crossmatch tests, suggesting potential application for crossmatch prediction. Moreover, while the utility of pre-transplant complement detection, at least in crossmatch-negative transplant recipients, is controversially discussed, a series of studies have shown that the appearance of post-transplant complement-fixing DSA may be associated with C4d deposition in transplant capillaries and a particular risk of graft failure. ConclusionsThe independent value of modified single antigen bead assays, as compared to a careful analysis of standard IgG detection, which may be affected considerably by complement dependent artifacts, needs to be clarified. Whether they have the potential to improve the predictive accuracy of our current diagnostic repertoire warrants further study.

10: EDTA-Treated Serum Unmasks Complement-Mediated Prozone Inhibition

The Luminex-based class I and II human leukocyte antigen (HLA) antibody testing is widely used for organ transplantation. However, false negative results can occur due to prozone inhibition when complement binds to antigen-bound HLA antibodies, thereby blocking access of secondary antibodies. The addition of ethylenediaminetetraacetic acid (EDTA) to serum can decrease complement-mediated prozone effect by chelating calcium needed for C1q assembly. The aim of this …

Validation and application of a canine-specific automated high-sensitivity C-reactive protein assay.

Measurement of low concentrations of C-reactive protein (CRP) in dogs has previously been performed with nonautomated assays. The aim of this study was to validate an automated high-sensitivity CRP (hsCRP) assay, developed by modifying a routinely used canine-specific immunoturbidimetric CRP test (cCRP). Imprecision, linearity under dilution, limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ) were determined for the hsCRP test, as well as the presence of prozone effect and interferences. The imprecision, measured as intra-assay variation, was ≤2.7%. The assay was acceptably linear under dilution. An analytically relevant prozone effect was present for samples with CRP concentration >150 mg/L, and there were mild interferences from hemolysis and lipemia. The LOB, LOD, and LOQ were 0.10 mg/L, 0.22 mg/L, and 0.50 mg/L, respectively. A method comparison study with a canine-specific enzyme-linked immunosorbent assay (ELISA) was performed, showing poor agreement between the hsCRP test and the ELISA. An additional aim of the study was to apply the hsCRP test to clinical research samples. Serum samples from 7 dogs undergoing ovariohysterectomy were collected pre- and postoperatively, and CRP was measured with both the cCRP and hsCRP assay. The expected postoperative increase in CRP was detected earlier with the hsCRP test, compared with the cCRP test. The hsCRP assay was further applied on samples from 6 lean and 9 overweight dogs. There was no significant difference in CRP concentration between the groups (P = 0.06). In conclusion, the hsCRP test had acceptable analytical performance, and the assay was successfully applied to clinical research samples.

OR09: KEEP IT COOL. A NOVEL INHIBITOR COMPLEX EXCLUSION (ICE) PROTOCOL FOR LABSCREEN THAT PREVENTS THE “PROZONE” EFFECT

Background An important limitation of the single antigen bead (SAB) assay is its sensitivity to the prozone effect, where high titer complement fixing HLA antibodies are undetected due to complement mediated interference with IgG-PE detection antibody. Strategies used to minimize the prozone effect include serum treatment with heat, DTT or EDTA, all inhibiting complement fixation. However, these treatments may compromise HLA antibody integrity and/or binding. Given that complement activation is markedly reduced at low temperatures we hypothesized that SAB testing performed at Methods 5 prozone positive and 5 prozone negative sera were tested in parallel using three protocols: (1) standard LABscreen (SL), (2) EDTA pre-treatment (ESL) and (3) inhibitor complex exclusion (ICE) protocol in which sera are incubated with SABs on ice instead of room temperature. Reactions were classified into inhibitor positive (IP), questionable (IQ) or negative (IN) based on SL to ESL MFI increase >10 K, 4 K-10 K and Results Parallel testing of 5 prozone positive sera with SL and ESL protocols identified 79 IP, 24 IQ and 857 IN rxns. 32/79 IP rxn had SL MFI Conclusion We have developed a novel ICE protocol, which prevents the prozone effect while maintaining excellent HLA antibody detection sensitivity. Importantly, ICE protocol avoids any potential interference with HLA antibody testing that may be caused by pre-treatment of sera.

OR38: CARFIZOMIB FOR REFRACTORY ANTIBODY MEDIATED REJECTION IN LUNG TRANSPLANTATION THE IMPACT ON DSA

Aim Antibody mediated rejection (AMR) after lung transplantation (LTx) is associated with early and severe chronic rejection and death. Conventional therapies consisting of plasma exchange and IVIG with and without Rituximab are partially effective. Newer therapies with proteasome inhibitors (Carfizomib (CFZ) and Bortezomib (BTZ)) that target both B and plasma cells may be more effective in removing the donor specific antibodies (DSA). CFZ, unlike BTZ, irreversibly inhibits the activity of 20S proteasome and results in less neuropathic side effects. Method We evaluated the impact of CFZ therapy in 7 LTx on DSA level and function and correlated with clinical outcome as measured by pulmonary function test. All 7 patients had DSA IgG specificity and titer measured by single antigen bead Luminex assay and DSA function of complement binding by C1q-screen at baseline and at 1 month and 3 months post-treatment. Results The specificity, strengths and C1q reactivity pre- and post-therapy is summarized in Table below. All 7 patients had class II DSA and 6/7 were DQ specific with C1q reactivity. 5/7 LTx responded to initial treatment with a drop in IgG DSA level, with negative C1q DSA reactivity ( 15,000 MFI the IgG DSA was inhibited (“prozone effect”), however IgG DSA significantly increased in the presence of EDTA (LTx 3, 5, 6, 7). Conclusion In this small cohort of LTx who mostly failed conventional therapies and have persistent DSA, CFZ based approach appears to be useful, initially. However, 5/7 LTx recovered their C1q DSA reactivity upon longer follow-up suggesting that one cycle of CFZ it may not be effective to impact the rebound of circulating DSA.

P026 : A BIOTIN/STREPTAVIDIN ENHANCED SINGLE ANTIGEN BEAD (SAB) METHOD IMPROVES SENSITIVITY AND OVERCOMES COMPLEMENT MEDIATED INHIBITION

Aim Microparticle-based, solid phase assays are the preferred method to detect and identify HLA antibodies employing both Luminex-based and standard flow cytometric approaches. However, discrepant results between platforms underscore the challenges associated with data interpretation. Therefore, the goal of this study was to develop a more sensitive and robust SAB assay. Methods A biotin-streptavidin system for HLA antibody detection (BIO-SAB) was compared to the standard SAB (STD) assay. A total of ten informative sera (five for Class I and five for Class II) were identified as being positive by FlowPRA[TM] and negative by STD-SAB. An additional ten sera (five for Class I and five for Class II) that had been previously identified to be EDTA-sensitive were also tested using both assays. Results Our findings revealed that the BIO-SAB method increased assay sensitivity approximately 4-fold over the conventional assay for Class I and Class II antibodies in all sera tested. Therefore, the BIO-SAB provided a better correlation with flow cytometry-based assays. Importantly, our modified assay was unaffected by complement-mediated inhibition (“prozone effect”). The BIO-SAB yielded results similar to EDTA-treated sera run by STD-SAB, thereby eliminating the need for pre-test serum manipulations. Conclusions Our modified SAB assay using a biotin/streptavidin detection system overcomes complement-mediated inhibition while improving detection of low level HLA antibodies, especially those recognizing public epitopes. Laboratories currently using SABs as the sole methodology for antibody detection would benefit from implementation of the BIO-SAB as it reduces the risk of missing a potentially clinically relevant antibody. For laboratories currently using both a SAB as well as a phenotype-based bead product, implementation of the BIO-SAB method would reduce the rate of discrepant results between methods as well as decrease the need for pre-testing manipulations. Implementation of this modified method to detect HLA antibodies will increase assay sensitivity and reduce the frequency of false negative results.

P032 : DSA IN THE ABSENCE OF KNOWN SENSITIZING EVENTS: WHAT TO DO?

Donor-specific HLA antibodies (DSA) carry a risk of antibody-mediated rejection (AMR) in kidney transplants. However, the relevance of DSA seen in unsensitized patients prior to transplant is unclear. We report a case of DSA in an unsensitized male investigated for potential live kidney transplantation. HLA typing of the patient and potential donor was performed by low/intermediate and high resolution SSP. HLA antibodies were screened on serial serum samples using Flow PRA beads, Luminex single antigen beads (SAB), Luminex phenotype beads, Flow phenotype beads and Flow single antigen beads. Crossmatching was performed by CDC and flow cytometry. A 41-year old male without prior sensitization history was investigated for a potential unrelated live kidney transplant (HLA mismatches: A1, B44, Cw4, DR7, DR13, DQ2, DQ6). Initial HLA antibody screening using Flow PRA beads appeared negative but Luminex SAB testing revealed antibodies to A25, A32, DR16 and mismatched DQ6 (5/6 beads including donor DQB1 ∗ 06:04/DQA1 ∗ 01:02-associated bead, MFIs ∼7000). Serum testing using Luminex phenotype beads, Flow phenotype beads and Flow single antigen beads all suggested reactivity against the mismatched DQ6 antigen. Interestingly, DTT-treated serum showed similar Luminex SAB reactivity patterns as untreated serum but fetal bovine serum-absorbed serum showed reactivity only to mismatched DQ6 beads and uncovered very weak class II reactivity on Flow PRA beads. The T- and B-cell CDC crossmatches were negative. Both T- and B-cell flow crossmatches were negative although the B-cell flow crossmatch showed a slight median channel shift of 70, albeit lower than our validated cut-off of 150. No prozone effect was observed on serum dilution. The clinical relevance of this HLA-DQ6 DSA is unclear in the context of negative crossmatches and the patient has not been transplanted yet. Still, despite no known sensitizing event, pretransplant DSA could lead to AMR, as recently suggested, and could represent an immunological risk.

When “Neat” Just Isnʼt Enough: Gaining Precision and Depth in HLA Antibody Data Interpretation Utilizing Titers and C1q Antibody Binding Assay.

BACKGROUND: Virtual crossmatches (VXM) are routinely performed utilizing solid-phase single antigen (SA) HLA antibody (Ab) assays to determine compatibility and induction and maintenance therapies for transplant recipients. SA tests are also a critical component of post-transplant monitoring for Ab mediated rejection (AMR). Neat, or undiluted, sera samples from highly sensitized patients (pts) are susceptible to prozone in these assays, which masks strong or high titer donor specific Ab (DSA). Titration and/or C1q-binding Ab (C1q) assays could alleviate these issues and add needed data to determine clinical risk from DSA. METHODS: We prospectively tested 47 neat and tittered samples from 34 highly sensitized pts who underwent kidney or heart transplant at our center, using commercially available SA-IgG and C1q kits. SA-IgG data was stratified into Strong (>10,000 MFI), Strong Moderate (5,001-10,000 MFI), Weak Moderate (2,501-5,000 MFI), Weak (1,000-2,500 MFI) and Negative Ab expression (< 1,000 MFI). C1q Ab expression was categorized as Strong (>10,000 MFI), Strong Moderate (5,001-10,000 MFI), Weak Moderate (1001-5,000 MFI), Weak (200-1000 MFI) and Negative (< 200 MFI). RESULTS: Prozone effect, as revealed by titration studies, was observed in 10/34 patients (11/47 tests) using neat sera SA-IgG tests. Prozone was most apparent at a titer of 1:16 on average; 0% of C1q results displayed prozone. Importantly, comparisons between neat MFI values and antibody strength, using titer evaluation, showed disconcordance in 19/34 patients. Specifically, 12/34 patients (15/47 tests) exhibited low neat MFI (1,000-5,000) while antibody titers were high (1:256->1:1024). Furthermore, 6/34 patients (7/47 tests) exhibited high neat MFI (>5,000) but the antibody strength per titer was low (1:1-1:32). CONCLUSION: Prozone effect was observed in 30% of patients in this cohort. Conflicting data between neat IgG MFI values and titers were observed in 56% of patients. While both titration and C1q studies alleviated this issue in 100% of the cohort, only titration studies informed a sense of avidity and affinity of Ab binding while maintaining high sensitivity, as only 9.4% of DSA tittering at or below 1:64 were detected by C1q. We conclude that titration studies provide valuable clinical information in accurately predicting VXM results and effectiveness of desensitization or of therapeutic modalities to resolve AMR. DISCLOSURES:Friedewald, J.: Grant/Research Support, Pfizer, Stockholder, TGI.

EFEITO PROZONA NO DIAGNÓSTICO DE SÍFILIS PELO MÉTODO VDRL: EXPERIÊNCIA DE UM SERVIÇO DE REFERÊNCIA NO SUL DO BRASIL

Backgound and Objectives: Syphilis is a systemic infectious disease caused by the spirochete Treponema pallidum. VDRL (Venereal Diseases Research Laboratory) test is the most often used laboratory method for syphilis diagnosis. The prozone effect may occur in 1% to 2% of patients, especially at late stages of syphilis and during pregnancy. The objective of this study was to evaluate the frequency of this effect in analyses performed at Laboratorio Regional de Santa Cruz do Sul do Laboratorio Central do Rio Grande do Sul, from April to May 2013. Methods: A descriptive study was performed, in which 2018 samples were analyzed by the VDRL method and subsequently, low titers (≤ 1:4) were confirmed by the immunoblot method. Results: A total of 68 (3.37%) samples were positive, of which 49 (72.1%) belonging to women and of these, 38 (77.5%) pregnant women. The VDRL method showed no prozone effect. The 32 samples with titers ≤ 1:4 were confirmed by the treponemal method, resulting in 6 false-positive results, of which 4 were observed in pregnant women. Conclusion: Although not prozone effect was observed in this study, it was concluded that the VDRL method results must be evaluated individually, with the possibility of confirmation through the treponemal test.

Complement Component C3 Activation

Luminex-based anti-HLA IgG detection on single-antigen flow beads (SAFB) represents a valuable tool for characterization of allosensitization patterns. Assay interpretation, however, may be impeded by false-low test results caused by the prozone effect. Recent experimental data have related this artifact to direct blockade of IgG detection by complement component C1 as a possible candidate mechanism. To dissect a causative role of C1 complex formation and subsequent steps of classical complement activation, native or modified sera obtained from transplant candidates with HLA class I sensitization were evaluated applying SAFB-based IgG, IgM, C1q, C1r, C1s, or C4 and C3 split product detection, respectively. Evaluating a total of 1,164 single-antigen reactions, serum dilution (1:10) revealed an 11% incidence of the prozone effect as defined by a greater than 100% increase in IgG mean fluorescence intensity. Prozoning was found to be related to the amount of antibody-triggered C1q, C4 or C3 split product deposition, and was eliminated by serum modifications affecting the integrity of the C1 complex (dithiotreitol, ethylenediaminetetraacetic acid, heat inactivation). Remarkably, the same effect was achieved without C1 disintegration, either by serum treatment with methylamine to block C4 and C3 split product binding or by cobra venom factor to trigger C3 consumption. Our results reinforce a central role of C1 as a trigger of prozoning. However, we provide evidence that C1 may exert its effects only indirectly, namely via inducing C3 fragment deposition, which by coating of the bead surface may block the binding of IgG detection reagents.

Combinatorial Inhibition in PDZ Containing Scaffold Proteins

By binding multiple ligands simultaneously, scaffold proteins increase the efficiency of signal transduction reactions and organize the flow of molecular information at cellular interfaces. Mathematical models predict that, unlike enzymes, scaffold proteins become self-inhibitory at high concentrations due to sequestration of ligands into separate, unproductive multi-protein complexes. This combinatorial inhibition (or prozone effect) suggests that there is an optimal scaffold concentration for the formation of specific multi-protein complexes, and that scaffold concentration could influence which complexes are formed and which signaling pathways are activated.

Brief Report: Spuriously Low Serum IgG4 Concentrations Caused by the Prozone Phenomenon in Patients With IgG4‐Related Disease

To determine the frequency of the prozone effect in patients with IgG4-related disease (IgG4-RD). After identifying the prozone effect in an index patient with IgG4-RD, we examined additional samples to determine the frequency of this phenomenon. Thirty-eight serum samples obtained from patients with IgG4-RD whose results had been reported previously were retested. The serum IgG4 concentrations determined by this repeat analysis were compared with the originally reported values. In 10 (26%) of 38 patients, the originally reported IgG4 values were falsely low; the prozone effect was identified in each of these 10 samples. Correction of the prozone effect by sample dilution led to revision of the mean serum IgG4 concentration in the 10 samples, from 26 mg/dl to 2,008 mg/dl (normal range 2.4-121 mg/dl). All 10 patients whose samples were affected by the prozone effect had active IgG4-RD. Failure to detect the elevated serum IgG4 concentrations had a direct impact on the decision not to institute treatment in these patients. The prozone effect may lead to major underestimations of IgG4 concentrations in patients with IgG4-RD and offers a potential explanation for the poor correlation observed between disease activity and serum IgG4 levels in some patients. This phenomenon should be considered if the serum IgG4 measurement appears discordant with the clinicopathologic diagnosis and the clinical assessment of disease activity.

Comprehensive Assessment and Standardization of Solid Phase Multiplex-Bead Arrays for the Detection of Antibodies to HLA-Drilling Down on Key Sources of Variation

To the Editor: We appreciate the opportunity to respond to the letter by Dr. Maillard and Dr. Mariat (1Maillard N Mariat C. Solid-phase bead-based assays limitations are not restricted to interlaboratory variability.Am J Transplant. 2013; 13: 3049Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). They raise three questions regarding our recent article focusing on inter-laboratory standardization of solid phase multiplex-bead arrays to detect antibodies to HLA within the framework of the Clinical Trials in Organ Transplantation (2Reed EF, Rao P, Zhang Z, et al. Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA. Am J Transplant 13: 1859–1870.Google Scholar). Our article provides insights into the key sources of variability in commercially available solid phase HLA antibody testing kits. Importantly, our study demonstrates that standardization of reagents and protocols significantly reduces assay variance. Utilization of a global normalization algorithm further reduced median fluorescence intensity (MFI) variations in the protocol, thereby improving comparison of data across laboratories. Dr. Maillard and Dr. Mariat comment that our study did not address the prozone effect. Although they raise an important point, we do not believe the prozone effect impacted our estimates of inter-laboratory variance, since all participants adopted a standardized protocol, used the same reagents and tested a constant volume of alloantisera. We do agree with Dr. Maillard and Dr. Mariat that adding dithiothreitol, running dilutions or employing other methods to explore potential prozone/interfering factors is worthy of systematic investigation. Dr. Maillard and Dr. Mariat correctly point out that the %CV decreases within higher MFI strata. Although they indicate this finding is presented in the Bland–Altman plots (2, figure 5), it is actually illustrated in figure 3 of our article, which shows the variation among seven centers across distinct MFI strata. Dr. Maillard and Dr. Mariat suggest that the sudden amelioration in %CV within higher MFI strata is due to saturation of the beads with antibodies. However, as we clearly showed, the decline in %CV begins at 1000 MFI, well below a saturation dosage (<10 000 MFI), which indicates saturation is not the primary reason to explain this result. Their third point questions the impact of intra-laboratory variability on results and whether the improvement in %CV was due to a reduction in variance within an individual laboratory or between laboratories. Since it is standard of care that clinical laboratories utilize a standard operating procedure for HLA antibody testing, we expect the major cause of assay variance is lot-to-lot differences in test kits. Although we did not specifically address intra-laboratory variability in our report, each data point shown in the Bland–Altman plot (2, figure 5) can be converted into a pseudo “intra-laboratory” %CV [i.e. |ΔMFI|/(2×avgMFI)] representing the variation when a lab repeats the test of same sample and bead across two lots of single antigen kits. The median intra-laboratory %CV was 19%, and boxplots demonstrate a decline with increasing MFI range within each center and overall (Figure 1). On average, the intra-laboratory %CV was less than our reported inter-laboratory %CV (∼25%). Nonetheless, we acknowledge that other sources of variation in the aspects of the assay can certainly contribute to intra-laboratory variability and that each laboratory needs to address these concerns. We anticipate that both inter- and intra-laboratory variance will decrease with the implementation of standardized testing protocols and the increasing availability of uniform lots of reagents. This research was performed as part of an American Recovery and Reinvestment (ARRA) funded project under Award Number U0163594 (to P. Heeger), from the National Institute of Allergy and Infectious Diseases. The work was carried out by members of the Clinical Trials in Organ Transplantation (CTOT) and Clinical Trials in Organ Transplantation in Children (CTOT-C) consortia. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

37-P

The introduction of Luminex-Single Antigen bead (LSA) assay significantly improved identification of HLA antibodies in sera from highly sensitized transplant candidates. Variants of this assay allowed to detect IgM antibodies and complement-fixing antibodies. Recently, the LSA assay was shown to be prone to the complement-mediated prozone effect leading to false-negative results. In this study we performed a comprehensive analysis of the HLA class I and class II antibodies present in sera from 16 highly sensitized kidney transplant candidates (%FlowPRA class I and II: 96 ± 4 and 95 ± 5 respectively) using four variants of the LSA technique: IgG-LSA, IgM-LSA, C1q-LSA and also EDTA-IgG-LSA to overcome the complement-mediated prozone effect. In 61% of EDTA-IgG-LSA tests we evidenced a strong increment of several antibody row-value (13,709 ± 5,863) respect to IgG-LSA results. This method was also able to reveal additional HLA class I and II antibodies (A2, A25, A68, A69, B42, B57, B59, B67, B77,DR17, DQ7, DQA1∗05:01) in 33% of the performed tests. Correlating EDTA-IgG-LSA and C1q-LSA results we found that all but one sera contained both complement-fixing and non complement-fixing antibodies; in the remaining serum we only detected anti-DP antibodies unable to fix complement. In one case, EDTA-IgG-LSA assay revealed the presence of a wide antibody pattern (anti-DQ2, -DQ4, -DQ7, -DQ8, -DQ9) specific for 84-87QLEL/89-90TT epitopes while C1q-LSA assay identified a similar but smaller antibody pattern (anti-DQ7, -DQ8, -DQ9) due to the recognition of 55P epitope. In 5 sera, IgM-LSA assay was able to detect HLA antibodies not revealed by IgG-LSA technique; unexpectedly several of these antibodies were HLA-Cw specific. In conclusion our findings evidenced the importance to make use of all LSA potentiality for characterization of HLA pre-sensitization status of transplant candidates in order to assess the real immunological risk of a potential kidney transplant.

Secondary syphilis and suspected child abuse

A 38-year-old Hispanic woman presented with a widespread papulosquamous eruption with accentuation on the palms and soles (Fig 1, A). Travel history was remarkable for a recent trip to her native country (Dominican Republic). Her 13-year-old son, who accompanied her on the trip, developed a similar rash after their return (Fig 1, B). In addition, the patient noted that her boyfriend, whom she was visiting in the Dominican Republic, had a rash when she arrived. The rapid plasma reagin (RPR) test produced negative findings whereas the skin biopsy specimen from the patient’s foot showed a psoriasiform lichenoid dermatitis teeming with spirochetes highlighted by a Steiner stain. After discussion with the laboratory about the RPR results, serial dilutions proved positive (prozone effect). At follow-up 4 days later, the son was evaluated for his rash. As expected, the patient’s fluorescent treponemal antibody absorption (FTA-ABS) test result was positive, but the son’s test finding was only minimally reactive.

Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola

Mice experimentally infected with a pathogenic strain of Leptospira interrogans serovar Canicola produced false negative results (prozone effect) in a microscopic agglutination test (MAT). This prozone effect occurred in several serum samples collected at different post-infection times, but it was more prominent in samples collected from seven-42 days post-infection and for 1:50 and 1:100 sample dilutions. This phenomenon was correlated with increased antibody titres in the early post-infection phase. While prozone effects are often observed in serological agglutination assays for the diagnosis of animal brucellosis and human syphilis, they are not widely reported in leptospirosis MATs.

1026-LB

To investigate the case of a potential Living Donor Renal Transplant with Negative CDC-AHG and Positive Flow cross match with diluted serum sample having high to moderate levels of multiple Class I and Class II DSAs. Both Patient and living related donor were HLAtyped for A, B, C, DR, DQ, DP by reverse SSOP-PCR.ClassI and ClassII HLA specific antibodies were determined by Luminex based single antigen bead assay. Serum samples before and after plasmapheresis/IVIG were tested for antibodies with and without OTTtreatment. One of the current serum samples was also tested for HLA-Specific IgM antibodies, and Clq binding antibodies. T and Bcell cross matches were performed on pronase treated cells by Flow cytometry using serum samples with and without OTTtreatment. The CDCAHG assay was performed on serum samples with and without OTT treatment. The patient consistently had both Class I and Class II DSAs against A74, Cw2, Cw4, DRl3, DR1S, DRS1, and DRS2 on serum samples collected in March 2012, and July 2012. After 2 rounds of plasmapheresis and IVIG treatment in the last week of July 2012, the DSA levels remained high, although the MFI values of some of the DSAs dropped insignificantly. The flow cytometry cross match was negative with untreated serum before plasmapheresis and was positive on NHS mixed serum (which is a 1:2 dilution), and in serum diluted 1:2 using PBSindicating a slight prozone effect. The CDC-AHG and CDCAmos cross matches with T and B cells were positive on untreated pre-plasmapheresis serum sample. However, after Dn treatment, the CDC-AHG and CDC-Amos cross matches were negative for both T and B cells indicating the CDC reactivity in untreated serum sample was due to IgM antibodies. It is to be noted that this same serum sample had multiple Class I and Class II DSAs detected by the Luminex SAB assay using anti-lgG as secondary antibodies. In order to determine the complement activating function of these DSAs, the pre-plasmapheresis serum sample was tested by C1qbinding assay and it turns out that there were no DSAs that reacted in the C1q binding assay. We also tested this pre-plasmapheresis serum for IgM class of HLA Class I & II antibodies, and only 4 Class I and 2 Class II antibodies were identified-none were DSAs; however the IgM class of HLA-A23 antibody could have clones that might react with the 9p public Epitope on the Donor’s HLA-A 2. And the Cw1 antibody could cross react with the donor’s Cw4 which might have contributed for the allo CDCT and B cell cross match reaction with untreated serum. Two serum samples were tested after plasmapheresis for DSAs, T and B cell flow and CDC-AHG cross match and the reaction pattern remained the almost the same except that the Flow cytometry cross match was positive on undiluted serum sample as well and CDC-AHG was positive with B cells using untreated serum, and negative when tested with Dn treated serum. In general, it appears that the patient has high levels of DSAs likely to be of the non-complement binding or weakly complement binding class of IgG antibodies namely, IgG2/4 subtypes. The Clinical implications of these indings will be discussed.

1019-LB

Aim Single antigen reagent panels differ in the makeup of HLA alleles offered for testing. Additional alleles (2 HLADR, 7 HLA-DQ) were added for the newest lot of Gen-Probe MHC Class II reagents in an effort to improve diversity of the panel and increase the ability to interchange between commercial products. However, an analysis of the correlation between single antigen reagents is necessary before results may be compared. Methods Twenty-two sera samples were tested in parallel with Gen-Probe (GP; Lot 03022V) and One Lambda (OL; Lot 9) single antigen MHC Class II reagents. GP assay requires addition of 40 μl bead mix + 10 μl sera, and the OL assay requires 5 μl bead mix + 20 μl sera, while the absolute number of beads is similar between assays. Murine monoclonal antibodies against HLA-DR, -DQ, and -DP with a concentration capable of saturating reagents were used to compare antigen density of GP and OL reagents. GP Background Corrected Mean (BCM) value and OL Normalized value in mean fluorescence intensity (MFI) from antigen density experiments were analyzed by paired student’s t-test. Correlation analysis (Pearson r) was performed on GP BCM and OL Normalized values between beads with equivalent molecular antigens (50 total, 27 HLA-DR, 13 HLA-DQ, and 10 HLA-DP) as well as on titer analysis (neat, 1:16, 1:256, and 1:1024) of 5 sera samples. Results Antigen density was significantly greater on GP reagents versus OL reagents with mean strength of saturating murine monoclonal antibodies being 8,080 ± 2,257 MFI versus 2,150 ± 516 MFI for HLA-DR, 9,955 ± 2,866 MFI versus 5,175 ± 1,256 MFI for HLA- DQ, and 11,084 ± 1,615 MFI versus 3,291 ± 553 MMF for HLA-DP (p Conclusions Both the significantly higher density of HLA antigens on the GP beads and a dilution effect introduced by the difference in serum to bead-volume ratio may contribute to the lower correlation observed in this study. This is supported in part by the higher prozone effect observed with OL HLA-DQ specificities but not in GP, and the improved correlation observed with titers. Further investigation is warranted to determine correlation between GP and OL reagents.

Evaluation of the malaria rapid diagnostic test SDFK90: detection of both PfHRP2 and Pf-pLDH

BackgroundRapid diagnosis of Plasmodium falciparum infections is important because of the potentially fatal complications. SDFK90 is a recently marketed malaria rapid diagnostic test (RDT) targeting both histidine-rich protein 2 (PfHRP2) and P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH). The present study evaluated its diagnostic accuracy.MethodsSDFK90 was tested against a panel of stored whole blood samples (n= 591) obtained from international travellers suspected of malaria, including the four human Plasmodium species and Plasmodium negative samples. Microscopy was used as a reference method, corrected by PCR for species diagnosis. In addition, SDFK90 was challenged against 59 P. falciparum samples with parasite density ≥4% to assess the prozone effect (no or weak visible line on initial testing and a higher intensity upon 10-fold dilution).ResultsOverall sensitivity for the detection of P. falciparum was 98.5% and reached 99.3% at parasite densities >100/μl. There were significantly more PfHRP2 lines visible compared to Pf-pLDH (97.3% vs 86.9%), which was mainly absent at parasite densities <100/μl. Specificity of SDFK90 was 98.8%. No lot-to-lot variability was observed (p = 1.00) and test results were reproducible. A prozone effect was seen for the PfHRP2 line in 14/59 (23.7%) P. falciparum samples tested, but not for the Pf-pLDH line. Few minor shortcomings were observed in the kit’s packaging and information insert.ConclusionsSDFK90 performed excellent for P. falciparum diagnosis. The combination of PfHRP2 and Pf-pLDH ensures a low detection threshold and counters potential problems of PfHRP2 detection such as gene deletions and the prozone effect.