Spherotech-EDTA combined serum treatment reduces background more effectively as compared to One Lambda Adsorb Out™ and LIFECODES Serum Cleaner in Luminex-based solid-phase immunoassays for HLA antibody detection.

Spherotech (SPT) microparticles capture non-specific binding materials present in test serum, and EDTA removes the so called" prozone effect". This study presents a novel approach of combined SPT-EDTA serum treatment prior to Luminex HLA antibody testing to remove high background, and prozone effect in a single step process, and compared the efficacy of SPT-EDTA serum pre-treatment with AdsorbOut (ADS) and Serum Cleaner (SC) to reduce background in solid phase immunoassays (SPI). A total of 21 serum samples with a history of elevated negative control (NC) values ≥500, and 20 samples with normal NC values were included to assess the potential adverse effects. A problem of high background was noted in 25% of our samples in SPI. We observed 80% effectiveness in reducing NC values <500 with SPT-EDTA serum pre-treatment compared to 72%, and 67% for ADS and SC-treated sera, respectively. Interestingly, we found a strong correlation in antibody-binding levels between SPT versus ADS; and ADS versus SC-treated sera for both phenotype and single antigen bead assays (p < 0.001). No adverse effect was noted on NC, positive control (PC) values, PC/NC ratios in the upfront use of SPT-EDTA as compared to EDTA alone. Our data revealed that combined SPT-EDTA treated sera is more effective than ADS, and SC in reducing high background in SPI. Taken together, SPT-EDTA serum treatment prior to Luminex HLA Ab testing is cost-effective, our laboratory saves nearly 30% of the annual total cost for Ab testing and improved test turnaround time by two business days.

Defects and countermeasures in laboratory diagnosis of rare IgE multiple myeloma.

BACKGROUND: IgE multiple myeloma (MM) is a rare subtype of MM that is easily misdiagnosed. We report a rare case of IgE-MM and investigate the application of the SLiM-CRAB criteria to screen for high-risk smoldering MM (SMM) patients, so as to summarize the causes and methods used to prevent missed diagnosis or misdiagnosis in IgE-MM. METHODS: The serum monoclonal protein (M-protein) classification and IgE quantification was performed and sent to several individual institutions. The results were collected and the causes of IgE detection defects were analyzed. RESULTS: Upon admission to our hospital, the patient's serum free kappa light chain was 1069.9 mg/L, free lambda light chain was 9.2 mg/L, and free kappa/lambda ratio was 115.9, which met the SLiM criteria, but without CRAB features. Immunofixation electrophoresis (IF) showed "M-like protein aggregation bands" in all lanes. After pretreatment with 1% ß-mercaptoethanol to depolymerize the aggregation of monoclonal protein, the "M-like protein aggregation bands disappeared. The other five institutions did not provide the correct typing results. The quantification of serum IgE was as high as 2.06 × 107 IU/mL, whereas 7 other testing institutions reported IgE levels ranging from 1.0 to 1100 IU/mL. CONCLUSION: High-risk biomarkers in SLiM criteria can achieve good therapeutic effects in rare IgE-MM patients. Serum immunofixation performed without antisera against IgE, insufficient identification of the lytic bands produced by high macromolecule aggregation in IF, and the absence of a prozone effect avoidance procedure during IgE quantitative detection are the primary causes of missed diagnosis or misdiagnosis in patients with IgE-MM.

Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays.

BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is "prozone effect," which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. MATERIALS AND METHODS: In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called "positive concoction" in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. RESULTS: Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. CONCLUSION: In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.

Leukoencephalopathy and cerebral edema as the presenting manifestations of SLE in an ANA-negative adolescent female: a case report and review of literature.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease with various clinical manifestations involving multiple organ systems. Neuropsychiatric manifestations of SLE have been associated with increased morbidity and mortality, thus it is important to recognize and diagnose the disease entity and treat early. When neuropsychiatric symptoms are involved, typically there are many other systemic features to aid in the diagnosis of SLE. Many autoantibodies have been discovered and are used to help diagnose SLE. The antibody present in most cases of pediatric SLE, as well as in many other rheumatic diseases, is the nonspecific antinuclear antibody (ANA). The ANA is a commonly used screening tool by primary care physicians when evaluating a patient with a possible rheumatic disorder. However, a small subset of SLE patients, 1-5%, present with a negative ANA, and it is important to keep SLE on the differential diagnosis in specific instances when a thorough infectious, metabolic and neurological workup has been completed and proven to be inconclusive. CASE PRESENTATION: This case involves a Hispanic adolescent female with a negative ANA who presented with diffuse cerebral edema secondary to leukoencephalopathy due to SLE with central nervous system involvement. She was normotensive on presentation and relatively symptom free aside from headache. She had an extensive workup while inpatient involving metabolic, infectious disease, rheumatology, and neurology prior to obtaining the diagnosis of SLE. She was treated with cyclophosphamide and rituximab with appropriate disease response. CONCLUSIONS: A review of the literature revealed 12 cases with SLE presenting with or developing diffuse cerebral edema and/or leukoencephalopathy. Our patient's case differs in that she was also ANA negative despite other autoantibody positivity. While she did have low complements and transient leukopenia, she did not present with other signs of organ involvement, which made the diagnosis of SLE with neuropsychiatric involvement quite challenging. We discuss the importance of keeping SLE on the differential diagnosis despite a negative ANA in complex cases after thorough workup has been unrevealing, and to consider initial screening with not only the ANA but also dsDNA and complements to avoid missed diagnoses.

"False negative" CSF cryptococcal antigen with clinical meningitis: Case reports and review of literature.

There is an increasing recognition of patients presenting with cryptococcal meningitis despite having a negative CSF cryptococcal antigen (CrAg). In this report, we describe three cases of patients with advanced immunosuppression who presented to hospital with "false negative" CSF cryptococcal antigen, two of whom had a positive fungal culture. We describe the challenge of CSF-CrAg negative cryptococcal meningitis and explore ways to overcome this challenge using newer diagnostic techniques.

Falsely decreased thyroglobulin levels in a patient with differentiated thyroid carcinoma.

We describe a case of a patient with metastasized differentiated thyroid carcinoma who was treated with total thyroidectomy followed-up by radioactive iodine treatment. During treatment and follow-up the thyroglobulin levels were assayed which surprisingly did not match the clinical condition. An analytical flaw was suspected. Re-analysis in the laboratory showed the presence of a high dose hook effect (HDH), resulting in falsely low Tg levels. This case shows that HDH effects in immunoassays, like the thyroglobulin assay, still exist in daily practice. Discordance between laboratory results and clinical condition underlines the importance of short lines of communication between clinical chemists and medical doctors.

False-negative result of serum cryptococcal antigen lateral flow assay in an HIV-infected patient with culture-proven cryptococcaemia

The detection of cryptococcal capsular antigen (CrAg) is very sensitive and specific, however false-negative results have been reported, mostly in cerebrospinal fluid. We report the case of an HIV-infected patient with CD4=42 cells/mL, asthenic, negative serum CrAg lateral flow assay (LFA) and culture-proven cryptococcaemia. Despite the high accuracy of LFA, false-negative result is possible. Careful clinical evaluation and close follow-up are relevant

False-negative result of serum cryptococcal antigen lateral flow assay in an HIV-infected patient with culture-proven cryptococcaemia.

The detection of cryptococcal capsular antigen (CrAg) is very sensitive and specific, however false-negative results have been reported, mostly in cerebrospinal fluid. We report the case of an HIV-infected patient with CD4 = 42 cells/mL, asthenic, negative serum CrAg lateral flow assay (LFA) and culture-proven cryptococcaemia. Despite the high accuracy of LFA, false-negative result is possible. Careful clinical evaluation and close follow-up are relevant.

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody Testing.

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

The ABCs (DRDQDPs) of the prozone effect in single antigen bead HLA antibody testing: Lessons from our highly sensitized patients.

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ±â€¯EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody Testing

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

Prozone effect on indirect immunofluorescence assay for anti-nuclear antibody testing / 国际检验医学杂志

Objective To explore the influence of prozone effect on anti-nuclear antibodies (ANA) testing by indirect immunofluorescence assay (IIFA).Methods The samples with high titer of ANA (≥1∶1 000) were selected from 880fresh serum samples, and were subsequently diluted in 1∶100, 1∶1 000and 1∶10 000ratio.Prozone effect was defined as fluorescence intensity from 1∶1 000dilution was stronger than that from1∶100dilution.The samples with prozone effect were determined manually or by Sprinter XL and EUROPattern.The samples with prozone effect were further characterized by combinations of fluorescence patterns, fluorescence intensities and autoantibody specificities.Results A total of 880samples were tested.Importantly, 34samples displayed prozone effect (3.86％in total and 29.57％in samples with ANA≥1∶1 000).Interestingly, prozone effect was identified by manual detection as well as by Sprinter XL with similar fluorescence patterns and fluorescence intensities.Notably, EUROPattern can only select the central area for identification.Among all samples with prozone effect, 74.42％samples exhibited fluorescence intensities of≥1∶10 000.Speckled pattern was the most prevalent fluorescence patterns in samples with prozone effect (46.51％).In addition, anti-RNP antibodies (62.79％) were the most popular autoantibodies in samples with prozone effect, followed by anti-dsDNA antibodies (51.16％) and anti-SSA antibodies (51.16％).Conclusion Prozone effect was present in ANA testing, especially in samples with high titers, resulting in underestimating the titers.The study highlighted that special attention should be paid to the prozone effect in clinical practice.

Upfront dilution of ferritin samples to reduce hook effect, improve turnaround time and reduce costs.

INTRODUCTION: Sandwich immunoassays offer advantages in the clinical laboratory but can yield erroneously low results due to hook (prozone) effect, especially with analytes whose concentrations span several orders of magnitude such as ferritin. This study investigated a new approach to reduce the likelihood of hook effect in ferritin immunoassays by performing upfront, five-fold dilutions of all samples for ferritin analysis. The impact of this change on turnaround time and costs were also investigated. MATERIALS AND METHODS: Ferritin concentrations were analysed in routine clinical practice with and without upfront dilutions on Siemens Centaur® XP (Siemens Healthineers, Erlang, Germany) immunoanalysers. In addition, one month of baseline data (1026 results) were collected prior to implementing upfront dilutions and one month of data (1033 results) were collected after implementation. RESULTS: Without upfront dilutions, hook effect was observed in samples with ferritin concentrations as low as 86,028 µg/L. With upfront dilutions, samples with ferritin concentrations as high as 126,050 µg/L yielded values greater than the measurement interval and would have been diluted until an accurate value was obtained. The implementation of upfront dilution of ferritin samples led to a decrease in turnaround time from a median of 2 hours and 3 minutes to 1 hour and 18 minutes (P = 0.002). CONCLUSIONS: Implementation of upfront dilutions of all ferritin samples reduced the possibility of hook effect, improved turnaround time and saved the cost of performing additional dilutions.

False negative single antigen bead assay: Is it always an effect of prozone?

BACKGROUND: Bead based flow cytometry and Luminex play a major role in identification of alloantibodies in renal transplant work-up. Strong sensitization events may lead to prozone phenomenon that can affect single antigen bead (SAB) assay and result in false negativity. However, this can also be due to high titer of other blocking antibodies. While methods like, heat inactivation, C1 inhibitor, Ethylene diamine tetra-acetic-acid and Dithio threitol treatment can remove interfering antibodies of complement and IgM, these methods are not optimal if false negativity is due to prozone effect, which is high titer of antibodies alone. METHODS: We hereby present a case of a highly sensitized renal transplant recipient with 64% panel reactive antibody positivity (PRA) and a subsequent negative SAB assay. This paradoxical finding hinted at SAB being a false negative result and serial dilutions were used to perform further tests. RESULTS: Serum dilutions lead to positive flow based panel reactive antibody (PRA) and flow cytometry crossmatch (FCXM), with an increasing trend in FCXM. CONCLUSIONS: In highly sensitized patients serial dilution should be considered during a transplant work-up to avoid missing any underlying antibodies. Serum dilution can be used as first option to circumvent prozone. Also, interference of other antibodies should not be labeled as prozone effect.

Cooperative binding mitigates the high-dose hook effect.

BACKGROUND: The high-dose hook effect (also called prozone effect) refers to the observation that if a multivalent protein acts as a linker between two parts of a protein complex, then increasing the amount of linker protein in the mixture does not always increase the amount of fully formed complex. On the contrary, at a high enough concentration range the amount of fully formed complex actually decreases. It has been observed that allosterically regulated proteins seem less susceptible to this effect. The aim of this study was two-fold: First, to investigate the mathematical basis of how allostery mitigates the prozone effect. And second, to explore the consequences of allostery and the high-dose hook effect using the example of calmodulin, a calcium-sensing protein that regulates the switch between long-term potentiation and long-term depression in neurons. RESULTS: We use a combinatorial model of a "perfect linker protein" (with infinite binding affinity) to mathematically describe the hook effect and its behaviour under allosteric conditions. We show that allosteric regulation does indeed mitigate the high-dose hook effect. We then turn to calmodulin as a real-life example of an allosteric protein. Using kinetic simulations, we show that calmodulin is indeed subject to a hook effect. We also show that this effect is stronger in the presence of the allosteric activator Ca 2+/calmodulin-dependent kinase II (CaMKII), because it reduces the overall cooperativity of the calcium-calmodulin system. It follows that, surprisingly, there are conditions where increased amounts of allosteric activator actually decrease the activity of a protein. CONCLUSIONS: We show that cooperative binding can indeed act as a protective mechanism against the hook effect. This will have implications in vivo where the extent of cooperativity of a protein can be modulated, for instance, by allosteric activators or inhibitors. This can result in counterintuitive effects of decreased activity with increased concentrations of both the allosteric protein itself and its allosteric activators.

EDTA Treatment of Serum Unmasks Complement-Mediated Prozone Inhibition in Human Leukocyte Antigen Antibody Testing.

OBJECTIVES: Luminex-based single-antigen bead human leukocyte antigen (HLA) antibody testing is widely used to define HLA antibodies for transplant compatibility. False-negative results can occur with complement-mediated prozone inhibition. This study assessed the effect of EDTA on the assay background reactivity and fluctuations in antibody mean fluorescent intensity. METHODS: Serum specimens were retrospectively tested using Luminex-based single-antigen beads with and without EDTA. Treated and untreated serum samples were compared by two measures: changes in background reactivity and changes in HLA antibody strength after EDTA treatment. RESULTS: Ten pretransplant and 48 posttransplant specimens were identified: lung (22), heart (10), kidney (21), heart/lung (two), pancreas (one), small bowel (one), and liver (one). After EDTA treatment, weak antibodies (below 2,000 mean florescent intensity) demonstrated the largest fluctuations. Newly identified HLA antibodies were seen in 16% (8/49) of class I and 26% (15/57) of class II beads. EDTA treatment did not result in false-negative reactions compared with untreated serum. CONCLUSIONS: EDTA serum pretreatment mitigated complement-mediated prozone inhibition and improved accurate HLA antibody detection. The background reactivity and the false-negative rate of the assay appear unchanged.

Prozone Effect in the Diagnosis of Lupus Anticoagulant for the Lupus Anticoagulant-Hypoprothrombinemia Syndrome.

OBJECTIVES: The main clinical sequela of a lupus anticoagulant is increased thrombosis risk. However, bleeding due to lupus anticoagulant-hypoprothrombinemia syndrome is a rare but well-described manifestation of antiphospholipid syndrome. The association of acute acquired hypoprothrombinemia is caused by a lupus anticoagulant's specificity to prothrombin, which results in clearance of prothrombin and bleeding due to hypoprothrombinemia (usually <10% of normal). Severe life-threatening bleeding is most frequently reported in children with systemic lupus erythematosus or in healthy children after viral infection. In such cases, steroid therapy is usually effective in controlling the bleeding problems and improving prothrombin levels. METHODS: We report one pediatric patient with a lupus anticoagulant who had acute hemorrhagic diathesis. RESULTS: The diagnosis in this case was complicated by the presence of a prozone effect in lupus anticoagulant testing. The prozone effect (also known as hook effect) refers to situations where very high concentrations of antibody mask detection, typically in antigen-antibody reactions, which depend on visualization of agglutination. Decreasing the antibody/antigen ratio results in detectable antigen-antibody complexes. CONCLUSIONS: We report for the first time a variation on this theme in a patient with a lupus anticoagulant-type antiphospholipid antibody and hypoprothrombinemia, which corrected with immunosuppression and restoration of normal prothrombin levels.