Prozone Effect Research Articles

Nonclinical immunogenicity assessment of E3112, a recombinant human hepatocyte growth factor, and its impact on pharmacokinetics in rats and monkeys

E3112 is a recombinant human hepatocyte growth factor currently in development for the treatment of acute liver failure. The assessment of immunogenicity is crucial in the development of biotherapeutics. Consequently, a semi-quantitative assay of anti-drug antibody (ADA) was developed in rat and monkey serum using a ligand binding assay with electrochemiluminescence detection. A standard tiered approach was employed for the immunogenicity assessment, comprising a screening assay and a subsequent confirmatory assay. In the assay validation studies, selectivity, sensitivity, prozone effects, reproducibility, drug tolerance, and stability were evaluated. These assessments were conducted using a surrogate positive control of ADA. The accuracy and precision of the surrogate ADA were within ± 20 % and 20 %, respectively. The stability of ADA was also confirmed under a variety of conditions. The developed assays were successfully employed for the assessment of immunogenicity in rats and monkeys following the administration of a repeated dose of E3112. The administration of E3112 resulted in an increase in ADA levels, with higher levels observed in rats than in monkeys. Systemic exposures of E3112 in rats with higher ADA levels were lower than those with lower ADA, confirming the utility of nonclinical immunogenicity in interpreting pharmacokinetics and its inter-individual variability.

12568 “Unusual Presentation Of Pituitary Macroadenoma: Co-secretion Of GH And Prolactin With Prozone Phenomenon"

Abstract Disclosure: F. Lopez Maldonado: None. A. Rodriguez Gonzalez: None. A. Mayorga Leon: None. E. Leon Milan: None. Background: Plurihormonal pituitary adenomas (10-15% cases) are linked to acromegaly, especially in young patients. ∼8% are associated with MEN1. They produce growth hormone (GH), prolactin (PRL), and sometimes thyroid-stimulating hormone (TSH), causing clinical effects from GH and excess PRL. Most are large and invasive in ∼50% of cases. Case Report: A 37-year-old male with no significant medical history, exhibited monthly right temporal headaches, ipsilateral eye pain, nausea, and vomiting, that responded to analgesics and antibiotics. Two years later he presented increased headaches, fainting, and decreased vision in the right eye. Over the next four years, his headaches worsened, leading to complete vision loss in the right eye and compromised vision in the left eye. Evaluation by Internal Medicine revealed bradylalia, recent memory changes, a prominent supraorbital arch, pale optic disc with centered vessels in the right eye. Additionally, macroglossia, prognathism, and acromegaly-like changes in extremities were noted. Campimetry revealed left temporal hemianopsia. Hormonal profile: LH 0.04 mIU/mL (1.24 - 8.6 mIU/mL), FSH 0.21 mIU/mL (1.27 - 19.2 mIU/mL), TSH 4.78 mIU/mL (1.24 - 19.2 mIU/mL), Total T4 2.04 mcg/dL (4.5 - 10.9 mcg/dL), PRL 1.1 ng/mL (2.8 - 13.13 ng/mL) (at dilution 1:100) >20,000 ng/mL, Cortisol 0.903 mcg/dL (3.7 - 19.4 mcg/dL), IGF-1 1001 ng/mL (41 - 246 ng/mL), GH 12 ng/mL (0.03 - 2.47 ng/mL), Total Testosterone 0.13 ng/mL (1.6 - 8.7 ng/mL) MRI report: Invasive macroadenoma measuring 49x43x48 mm with partial inclusion of the right cavernous sinus and complete inclusion of the left cavernous sinus, bilateral carotid involvement with left predominance, involvement over the optic chiasm, pituitary stalk, floor of the sella turcica with migration towards the sphenoidal sinus. Diagnosis: Co-secreting PRL and GH pituitary macroadenoma, Secondary Hypogonadotropic Hypogonadism, Secondary Hypothyroidism, Secondary Adrenal Insufficiency. Treatment: Prednisone 5 mg PO QD, Cabergoline 0.5 mg PO QD 5/7 (MWFSS), Levothyroxine 100 mcg PO QD, Sostenon 240 mg/mL IM every 3 weeks. One week post-treatment initiation, the patient reported improvement.Follow-up labs showed normalized hormone levels. MRI revealed a 37x34x28 mm pituitary macroadenoma with bleeding and cystic changes. Surgery was deferred due to the favorable response to medical treatment. An annual MRI was performed, which showed a decrease in tumor size. By the seventh year, the tumor had reduced by 98% under the same treatment. Conclusion: Macroprolactinomas pose diagnostic challenges due to the prozone effect, requiring sample dilution for accurate diagnosis. Reflecting on the unexpected, this tumor co-secretes GH, which is uncommon; however, it responded effectively to medical treatment. Presentation: 6/3/2024

Nonclinical pharmacokinetics of E3112, a recombinant human hepatocyte growth factor, in rats and monkeys by a simple ELISA kit assay

E3112 is a recombinant human hepatocyte growth factor which is under development for the treatment of acute liver failure. Pharmacokinetics (PK) evaluation in experimental animals is important and thus a simple assay for the determination of E3112 in rat and monkey serum has been validated using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. E3112 in rat and monkey serum was quantifiable from 0.313 ng/mL to 15.0 ng/mL without prozone effects. Dilution integrity enabled accurate assay up to 500,000-fold dilution. Accuracy and precision were within the acceptance criteria. PK of E3112 was investigated after intravenous administration to rats and monkeys. PK of E3112 was similar between male and female animals in both species. Nonlinear PK of E3112 was observed in rats after intravenous bolus dose at 1–100 mg/kg while nonlinear PK was not significant in monkeys after intravenous infusion at 0.5–25 mg/kg. These findings suggest that the assay of E3112 in serum using a commercially available ELISA kit was validated and successfully applied to PK studies in rats and monkeys.

Combating Prozone Effects and Predicting the Dynamic Range of Naked-Eye Nanoplasmonic Biosensors through Capture Bioentity Optimization.

Accurately quantifying high analyte concentrations poses a challenge due to the common occurrence of the prozone or hook effect within sandwich assays utilized in plasmonic nanoparticle-based lateral flow devices (LFDs). As a result, LFDs are often underestimated compared to other biosensors with concerns surrounding their specificity and sensitivity toward the target analyte. To address this limitation, here we develop an analytical model capable of predicting the prozone effect and subsequently the dynamic range of the biosensor based on the concentration of the capture antibody. To support our model, we conduct a sandwich immunoassay to detect C-reactive protein (CRP) in a phosphate-buffered saline (PBS) buffer using an LFD. Within the experiment, we investigate the relationship between the CRP dynamic range and the prozone effect as a function of the capture antibody concentration, which is increased from 0.1 to 2 mg/mL. The experimental results, while supporting the developed analytical model, show that increasing the capture antibody concentration increases the dynamic range. The developed model therefore holds the potential to expand the measurable range and reduce costs associated with quantifying biomarkers in diverse diagnostic assays. This will ultimately allow LFDs to have better clinical significance before the prozone effect becomes dominant.

Cu(II)-induced magnetic resonance tuning and enhanced magnetic relaxation switching immunosensor for sensitive detection of chlorpyrifos and Salmonella

Magnetic relaxation switching (MRS) biosensors have been recognized as useful analytical tools for a range of targets; however, traditional MRS biosensors are limited by the “prozone effect”, resulting in a narrow linear range and low sensitivity. Herein, we proposed a paramagnetic Cu2+-induced magnetic resonance tuning (MRET) strategy, based on which Cu2+ ions and magnetic nanoparticles (MNPs) were adopted to construct a Cu–MNP-mediated MRS (Cu–M-MRS) immunosensor with Cu2+ ions acting as a quencher and MNPs as an enhancer. An Fe3O4@polydopamine-secondary antibody conjugate was prepared and used to correlate the amount of Cu2+ ions to the target concentration through an immunoassay. Based on the immunoreaction, the Cu–M-MRS immunosensor enabled the sensitive detection of chlorpyrifos (0.05 ng/mL, a 77-fold enhancement in sensitivity compared with the traditional MRS immunosensor) and Salmonella (50 CFU/mL). The proposed MRET strategy effectively improved the sensitivity and accuracy of the MRS immunosensor, offering a promising and versatile platform for food safety detection.

Concordance between APOE genotyping and proteotyping using the novel Lumipulse G Pan‐ApoE and Lumipulse G ApoE4 RUO assays

AbstractBackgroundThe APOE gene has three different alleles that encode for three different ApoE protein isoforms (APOE2, APOE3, and APOE4), resulting in 6 different phenotypes. Nucleic acid amplification test remains the gold standard for APOE genotyping, however quantification of the ApoE proteins using immunoassays could provide another piece of information, related to the expression levels of the proteins. In this regard, ApoE as a protein biomarker can be used in research to determine its value alone or as part of a panel of plasma biomarkers.MethodThe LUMIPULSE G System is a chemiluminescent enzyme immunoassay platform enabling fully automated processing of samples using ready‐to‐use immunoreaction cartridges. Time to result in these single use test cartridges takes about 30 minutes. The analytical performance of the newly developed Lumipulse G Pan‐ApoE and Lumipulse G ApoE4 RUO assays was determined. Parameters analyzed were precision, sensitivity, linearity, prozone and potential impact of interfering substances. Proof of concept of these novel assays was verified with a set of 230 genotyped plasma samples.ResultThe total within‐lab variability of both assays was ≤8% CV confirming high precision of the LUMIPULSE G system. Using low ApoE concentrated plasma samples, the observed LoD and LoQ (@20% CV) was 0.072 µg/mL (Pan‐ApoE) and 0.152 µg/mL (ApoE4). Linearity was shown across the assay range (0 – 100 µg/mL) for both assays. Neither prozone effect nor significant impact (recovery within 100±10%) of commonly tested endogenous substances (bilirubin, haemoglobin, low and high Protein content, and human anti‐mouse antibodies (HAMA)) was observed. Potential interference was observed for triglycerides and rheumatoid factor (RF) with observed recovery within 100±20%. ApoE proteotyping based on the novel assays resulted in a 99.8% concordance with APOE genotyping (1 discordant result). Moreover, applying the ratio ApoE4/Pan‐ApoE with the measurement values of respective assays, classified correctly between hetero‐ and homozygous E4 carrier status.ConclusionThe analytical performance studies demonstrate low variability and high sensitivity enabling measurement of pan‐ApoE and ApoE4 in plasma. Pan‐apoE and apoE4 quantification can offer relevant insights into the assessment of subjects at risk for AD or adverse events following anti‐amyloid treatments and should be studied further.

P‐IG‐24 | Immune‐Mediated Platelet Transfusion Refractoriness Complicated by Panagglutinin and Prozone Effect: A Case Report

P‐IG‐24 | Immune‐Mediated Platelet Transfusion Refractoriness Complicated by Panagglutinin and Prozone Effect: A Case Report

Development and validation of an automated assay for anti-drug-antibodies in rat serum.

The potential immunogenicity of therapeutic human and humanized monoclonal antibodies (mAb) is a significant concern, and so preclinical testing of therapeutic mAbs routinely includes assessment of anti-drug antibody (ADA) induction. Here, we report the development of automated screening and confirmatory bridging ELISAs for the detection of rat antibodies against DH1042, an engineered human mAb for the SARS-CoV-2 receptor-binding domain. The assays were evaluated for specificity, sensitivity, selectivity, absence of a prozone effect, linearity, intra- and inter- assay precision, and robustness, and found to be suitable for purpose. The assays were then used to evaluate anti-DH1042 antibodies in the sera of rats dosed with lipid-nanoparticle (LNP)-encapsulated mRNA encoding DH1042. Rats received two doses of 0.1, 0.4 or 0.6 mg/kg/dose LNP-mRNA 8 days apart. Twenty-one days after the second dose, 50-100% of rats had developed confirmed anti-DH1042 ADA depending on dose level. No animals in the control group developed anti-DH1042 ADA. These assays reflect new applications for a non-specialized laboratory automation platform, and the methodologies and approaches reported here provide a template that can be adapted for the automated detection and confirmation of ADA in preclinical testing of other biologics.

A-120 Performance Evaluation of Three Sentinel Diagnostics’ Assays on Roche Cobas c 303 Analytical Unit

Abstract Objective The aim of this study is to evaluate the analytical performances of the following Sentinel Diagnostics’ assays: Total Bile Acid Liquid, Copper, Aldolase, on Roche cobas c 303 analytical unit. Methods Performance evaluation included Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ), linearity, intra assay precision, inter assay precision, on board reagent stability, prozone effect, instrument correlation following the current CLSI guidelines protocols. Data were evaluated using Microsoft Excel statistical tool Analyse-it. Analytical Measuring Range (AMR) was defined through linearity and sensitivity. Results Summary of the analytical performances for all the assays tested is reported in table below. Conclusion All assays showed good results for all the parameters tested on Roche cobas c 303 analytical unit making these kits very suitable for a routine measurement of these analytes.

A-072 An All-Purpose Particle Enhanced Immunoturbidimetric Assay for the Comparable, Instrument-Independent Monitoring of Procalcitonin in the Management of Sepsis

Abstract Introduction Sepsis is a life-threatening organ dysfunction caused by a dysregulated host immune response to infection. It is a global health concern and a leading cause of death worldwide, affecting an estimate of 48.9 million people every year. Procalcitonin (PCT) revealed to be an indispensable parameter for the management of sepsis. However, recent analysis of EQA survey results suggested crucial deviations of PCT measurements in Germany and studies by the IFCC Working Group on standardization of PCT stated the need for a higher-order reference measurement procedure.Here we present the evaluation of the all-purpose particle enhanced immunoturbidimetric assay (PETIA) of DiaSys, which can be easily applied to every high-throughput clinical chemistry analyzer. The high diversity of applications and datasets match current diagnostic and regulative needs (IVDR). Procalcitonin FS enables the equivalent and instrument-independent monitoring of PCT in the management of sepsis. Methodology Evaluation of Procalcitonin FS was performed on seven clinical chemistry analyzer platforms (Jeol BioMajesty® JCA-BM6010/C, DiaSys respons®920 and respons®940, Roche cobas c501, Abbott Architect c8000 and Alinity c, Beckman Olympus AU680). Measurements were performed according to Clinical and Laboratory Standards Institute (CLSI) protocols. Results The onboard stability reaches 8 weeks (with chimney) on BioMajesty® JCA-BM6010/C, 20 days on respons®920 and respons®940, 21 weeks on cobas c501, 9 weeks on Architect, 12 weeks on Olympus AU680 and 13 weeks on Alinity c. The calibration stability is 4 weeks (with chimney) on BioMajesty® JCA-BM6010/C, 2 weeks (with chimney) on respons®920 and respons®940, 21 weeks on cobas c501, 3 weeks on Architect, 5 weeks on Olympus AU680 and 10 weeks on Alinity c. The intra-assay precision (CV%) ranges from ≤4.57% on respons®920 up to ≤7.19% on respons®940. The Total Precision (CV%) ranges from ≤3.99% on cobas c501 up to ≤9.62% on Olympus AU680. LOB ranges from 0.07 ng/mL on BioMajesty® JCA-BM6010/C up to 0.130 ng/mL on Olympus AU680. LOQ ranges from 0.20 ng/mL on Architect up to 0.277 ng/mL on Olympus AU680. The reagent displayed a linear recovery up to 50 ng/mL and no prozone effect was detected up to 1000 ng/mL on all seven analyzers. The Passing & Bablok regression vs the reference test showed an excellent comparability/alignment between the methods (slope ≤1.11, intercept ≤0.156 ng/mL, R ≥ 0.986). Conclusion Evaluation work proves that DiaSys PETIA reagent Procalcitonin FS shows excellent performance on all seven clinical chemistry analyzers. In addition, the high comparability between the seven analyzers enables the reliable and instrument-independent monitoring of PCT in the management of sepsis, and thus offering a solution to the current landscape of deviating PCT measurement results.

P100 HLA-Cw epitope analysis can resolve positive physical flow cytometry crossmatch in absence of donor-specific antibodies and prozone effect – A case study

P100 HLA-Cw epitope analysis can resolve positive physical flow cytometry crossmatch in absence of donor-specific antibodies and prozone effect – A case study

A Double-blind Randomized Comparative Phase I Study to Assess Biosimilarity and Immunogenicity of "Trastuzumab" (LLC "Mabscale", Russia) and Herceptin® (F. Hoffmann-La Roche Ltd., Switzerland) in Healthy Volunteers

Introduction. Trastuzumab is the first drug based on the monoclonal antibodies’ technology targeted to the neu oncogene expression product discovered in the middle 80-s – human epidermal growth receptor, HER2. After being approved trastuzumab had become the drug of choice for combine therapy of metastatic breast cancer (BC). This therapy had also allowed to improve patients’ 5-year survival rate dramatically, almost up to 90 % in some cases. Despite the fact that more than 10 biosimilars of trastuzumab are now in the pipeline around the world, including Russia, the development and registration of trastuzumab biosimilars still remain relevant.Aim. Aim of the study was to conduct the analytical part of the double-blind randomized comparative clinical trial for trastuzumab pharmacokinetics and safety assessment in healthy volunteers with subsequent biosimilarity evaluation of "Trastuzumab" (LLC "Mabscale", Russia) and Herceptin® (F. Hoffmann-La Roche Ltd., Switzerland).Materials and methods. 92 healthy volunteers, who fulfilled the inclusion/exclusion criteria, were enrolled to the study. Trastuzumab quantitation and anti-trastuzumab antibodies detection was performed using ELISA method with photometric detection. To support the clinical trial two different independent bioanalytical methods were validated.Results and discussion. Trastuzumab quantitation method in human blood serum was validated for selectivity, calibration curve and regression model, sensitivity (LLOQ), accuracy and precision, MRD, dilution linearity and stability. The method for anti-trastuzumab antibodies detection, that was previously described by the authors, was validated for cut-point, selectivity, sensitivity, prozone effect, drug tolerance, precision and stability (short-term and long-term). The validated methods were successfully applied to the study samples assay to perform the analytical part of the comparative study for trastuzumab pharmacokinetics and immunogenicity assessment. The obtained drug concentrations were used for PK-parameters and confidence interval calculations to estimate the biosimilarity of test and reference drug.Conclusion. The study results showed that test and reference drug are biosimilar, and moreover immunogenicity assessment showed no anti-trastuzumab antibodies in any samples of healthy volunteers.

Evolution of Antidrug Antibody Assays During the Development of Anti-Tissue Factor Pathway Inhibitor Monoclonal Antibody Marstacimab.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia.

Monitoring of Sotrovimab-Levels as Pre-Exposure Prophylaxis in Kidney Transplant Recipients Not Responding to SARS-CoV-2 Vaccines.

Background Sotrovimab, a monoclonal antibody against SARS-CoV-2, is used as a pre-exposition prophylaxis (PrEP) against COVID-19, but monitoring strategies using routine test systems have not been defined. Methods Twenty kidney transplant recipients without antibodies after vaccination received 500 mg Sotrovimab. Antibody levels were quantified over eight weeks using live-virus neutralization (BA1 and BA2), antibody binding assays (TrimericS, Elecsys, QuantiVAC) and surrogate virus neutralization tests (sVNTs; TECOmedical, cPass and NeutraLISA). Results Sotrovimab neutralized both Omicron subvariants (BA1 NT titer 90 (+-50) > BA2 NT titer 33 (+-15) one hour post infusion). Sotrovimab was measurable on all used immunoassays, although a prior 1:100 dilution was necessary for Elecsys due to a presumed prozone effect. The best correlation with live-virus neutralization titers was found for QuantiVAC and TrimericS, with a respective R2 of 0.65/0.59 and 0.76/0.57 against BA1/BA2. Elecsys showed an R2 of 0.56/0.54 for BA1/BA2, respectively. sVNT values increased after infusion but had only a poor correlation with live-virus neutralization titers (TECOmedical and cPass) or did not reach positivity thresholds (NeutraLISA). Conclusion Antibody measurements by the used immunoassays showed differences in antibody levels and only a limited correlation with neutralization capacity. We do not recommend sVNTs for monitoring SARS-CoV-2 neutralization by Sotrovimab.

Virtual and Reality: An Analysis of the UCLA Virtual Crossmatch Exchanges.

The "virtual" crossmatch (VXM) has become a critical tool to predict the compatibility between an organ donor and a potential recipient. Yet, nonstandardized laboratory practice can lead to variability in VXM interpretation. Therefore, UCLA's VXM Exchange survey was designed to understand factors that influence the variability of VXM prediction in the presence of HLA donor-specific antibody (DSA). Thirty-six donor blood samples and 72 HLA reference sera were sent to 35 participating laboratories to perform HLA antibody testing, flow crossmatch (FXM), and VXM from 2014 to 2019, consisting of 144 T/B-cell FXM pairs and 112 T/B-cell VXM pairs. In the FXM survey, 86% T-cell FXM and 84% B-cell FXM achieved >80% concordance among laboratories. In the VXM survey, 81% T-cell VXM and 80% VXM achieved >80% concordance. The concordance between FXM and VXM was 79% for T cell and 87% for B cell. The consensus between VXM and FXM was high with strong DSA. However, significant variability was observed in sera with (1) very high titer antibodies that exit prozone effect; (2) weak-to-moderate DSA, particularly in the presence of multiple weak DSAs; and (3) DSA against lowly expressed antigens. With the increasing use the VXM, standardization and continuous learning via exchange surveys will provide better understanding and quality controls for VXM to improve accuracy across all centers.

A Novel Approach for Modeling Biphasic Dose–Response Curves

Estimates of EC 50 1 from dose–response data play an important role in comparing drug potencies. When the sampling data of dose–response studies fail to follow a sigmoidal shaped curve, and the data display a biphasic property at higher dose levels where the response profile concaves and takes an inverted U-shape, this is known as the hook or prozone effect. To address this concern, some research investigators may pursue data removal. Others may choose to ignore the data shape and fit a model blindly. Unfortunately for both practices, the estimates of the fitting parameters, such as the EC 50, will be of poor quality and result in misleading inference. The authors propose the use of an empirical and novel extension of a sigmoid model to properly and effectively capture the information from all of the dose–response data, including that of the inverted U-shaped tail. Methods for using 3- and 4-parameter logistic models with examples, are discussed.

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.

Versatile performance edges of HBsAg Next assay in diagnosis and therapeutic monitoring of HBV infection.

HBsAg Next assay (HBsAgNx) claims improved detection of HBsAg. The aim was to investigate its performance in ascertaining HBsAg loss, ability to detect HBsAg in various phases of HBV infection, specificity and its amenability to in-house neutralization. Analytical sensitivity was investigated using NIBSC standard (3rd WHO-IS). For clinical performance, out of 91,962 samples tested for HBsAg (Qual-II), 512 samples consisting of 170 cases with evidence of HBsAg loss during treatment (n=116) and without treatment (n=54), acute-hepatitis B (n=90) and acute exacerbation of chronic-hepatitis B (n=41), acute-hepatitis A (n=24) and acute-hepatitis E (n=9) positive, HIV-1 positive (n=20), non-HBV, HAV and HEV related acute-hepatitis (n=81) and HBsAg prozone (n=14) as well as in-house neutralization (n=63) were included. The calculated limit of detection (LOD) was 0.004IU/mL. Of the 170 patients with apparent HBsAg loss, 18/116 (15.5%) among treated and 15/54 (27.7%) with spontaneous clearance were positive in HBsAgNx (p<0.0001). Additionally, it detected HBsAg in 12/95 (12.6%) and 6/34 (17.6%) patients who were HBV DNA negative in treatment experienced and spontaneous clearance groups respectively (p<0.001). The specificity of HBsAgNx was comparable to HBsAg Qual-II. The signal-intensity of HBsAgNx was significantly higher than HBsAg Qual-II across various phases of HBV infection and prozone samples. HBsAgNx significantly enhanced the accuracy of HBsAg detection without compromising the specificity in ascertaining HBsAg loss. The performance was superior in various phases of HBV infection including samples that exhibited prozone effect. Furthermore, it is amenable to cost-effective in-house neutralization to confirm low HBsAg levels.

Unexpected false-negative result in a traveller's malaria diagnosis.

A four-year-old traveller returned from Senegal with symptoms compatible with malaria. Plasmodium falciparum HRP-2 antigen detection was negative while panmalarial aldolase was positive. Microscopy confirmed the presence of P. falciparum trophozoites with parasitaemia of 16%. Discarding other causes of false-negative antigen detection, this is a rare case of prozone effect.

Validation of an automated immunoturbidimetric assay for feline serum amyloid A

BackgroundSerum Amyloid A (SAA) is a major acute phase protein in cats, increasing rapidly in response to various inflammatory diseases. An automated latex-enhanced immunoturbidimetric assay for human SAA (LZ-SAA, Eiken), previously validated for use in cats, has had further major modification (VET-SAA, Eiken) for specific use in veterinary diagnostic laboratories but has yet to be validated in cats.ResultsIntra-assay and inter-assay CVs for the VET-SAA assay ranged from 1.88–3.57% and 3.98–6.74%, respectively. Linearity under dilution was acceptable with no prozone effect observed. Limit of detection was 1.65 mg/L and limit of quantification was 6 mg/L. Haemoglobin and triglyceride showed no adverse interference, but bilirubin produced positive bias in samples with low SAA. Comparison with the LZ-SAA assay showed significant correlation with proportional bias increasing as SAA concentration increased, likely related to differing calibration standards. SAA was significantly higher in patients with inflammatory disease compared with non-inflammatory disease, and in patients with moderate to highly elevated α1-AGP compared with patients with normal α1-AGP. Improvement of the assay range may be required to fully evaluate differences between disease groups at low SAA levels. Based on ROC curve analysis, at a cut-off point of 20.1 mg/L the VET-SAA assay discriminated between inflammatory and non-inflammatory disease with sensitivity of 0.93 and specificity of 0.99.ConclusionsThe automated VET-SAA assay is a robust, precise, and accurate method for measurement of feline SAA which can clearly identify patients with inflammatory disease. It should be a valuable biomarker for use in feline medicine.