Use of pooled serum samples to assess herd disease status using commercially available ELISAs.

Pooled samples are used in veterinary and human medicine as a cost-effective approach to monitor disease prevalence. Nonetheless, there is limited information on the effect of pooling on test performance, and research is required to determine the appropriate number of samples which can be pooled. Therefore, this study aimed to evaluate the use of pooled serum samples as a herd-level surveillance tool for infectious production-limiting diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL) and Neospora caninum (NC), by investigating the maximum number of samples one can pool to identify one positive animal, using commercial antibody-detection ELISAs. Four positive field standards (PFS), one for each disease, were prepared by pooling highly positive herd-level samples diagnosed using commercially available ELISA tests. These PFS were used to simulate 18 pooled samples ranging from undiluted PFS to a dilution representing 1 positive in 1,000 animals using phosphate-buffered saline as diluent. A 1:10 dilution of the PFS resulted in positive results for IBR, BVD and EBL. Moreover, for IBR and BVD, results were still positive at 1:100 and 1:30 dilutions, respectively. However, for NC, a lower dilution (8:10) was required for a seropositive result. This study indicates that, at herd-level, the use of pooled serum is a useful strategy for monitoring infectious diseases (BVD, IBR and EBL) but not NC, using readily available diagnostic assays.

An updated protocol based on CLSI document C37 for preparation of off-the-clot serum from individual units for use alone or to prepare commutable pooled serum reference materials.

Manufacturers of in vitro diagnostic medical devices, clinical laboratories, research laboratories and calibration laboratories require commutable reference materials that can be used in the calibration hierarchies of medical laboratory measurement procedures used for human specimens to establish metrological traceability to higher order reference systems. Commutable materials are also useful in external quality assessment surveys. In order to achieve these goals, matrix-based reference materials with long-term stability, appropriate measurand concentrations and commutability with individual human specimens are required. The Clinical and Laboratory Standards Institute (CLSI) guideline C37-A (now archived) provided guidance to prepare commutable pooled serum reference materials for use in the calibration hierarchies of cholesterol measurement procedures. Experience using the C37-A guideline has identified a number of technical enhancements as well as applications to measurands other than cholesterol. This experience is incorporated into this updated protocol to ensure the procedure will continue to meet the needs of the medical laboratory. The updated protocol describes a procedure for preparing frozen human serum units or pools with minimal matrix alterations that are likely to be commutable with individual human serum samples. The protocol provides step-by-step guidance for the planning phase, collection of individual serum units, processing the units, qualifying the units for use in a pool and frozen storage of aliquots of pooled sera to manufacture frozen serum pools. Guidance on how to perform quality control of the final product and suggestions on documentation are also provided.

A retrospective evaluation of pooled serum ELISA testing in the frame of the French eradication program for infectious bovine rhinotracheitis.

Pooled serum testing using whole-virus indirect ELISA has been recently recognized as an official method for surveillance of bovine herpesvirus 1 (BoHV1) in cattle herds in Europe. In this study, a retrospective analysis of data from the French BoHV1 surveillance campaign 2018-2019, including 7434 BoHV1-free certified herds and 157 infected herds, was performed in order to evaluate the diagnostic specificity and sensitivity of two pooled serum indirect ELISAs (from IDEXX and IDVet), in comparison with individual testing by blocking ELISAs targeting the gB and gE proteins. Pooled serum testing showed a relative specificity higher than 97.5% and a detection rate of 100% since all gB+/gE+ samples were found in positive pools. At the herd level, no more than one false positive pool was observed in most of BoHV1-free certified herds, leading to a herd relative specificity of 85.1% and 86.0% for the IDEXX and IDVet pooled serum ELISAs, respectively. Among infected herds tested by pool sizes up to 10 sera (n = 122), 46% of herds were detected through pools of size 10 containing a single positive sample, 23% through pools of size 10 containing at least two positive samples, and 31% through pools of smaller sizes. A complementary study based on manually constituted pools revealed that at least one positive sample in 100% and 93.4% of herds could be detected individually by pools of size 10 with the IDEXX and IDVet ELISAs, respectively. However, pooled serum ELISAs were influenced by the level of individual reactivity, since pools composed of either one weak-positive sample or one gB+/gE- sample could yield negative results. Altogether, these results provided the first evidence that pooled serum testing (pool size up to 10) is a suitable strategy for surveillance of BoHV1-free cattle farms.

Porcine reproductive and respiratory syndrome virus RNA detection in tongue tips from dead animals.

The control of porcine reproductive and respiratory syndrome virus (PRRSV) hinges on monitoring and surveillance. The objective of this study was to assess PRRSV RNA detection by RT-PCR in tongue tips from dead suckling piglets compared to serum samples, processing fluids, and family oral fluids. Tongue tips and serum samples were collected from three PRRSV-positive breeding herd farms (farms A, B, and C) of three different age groups: newborns (<24 h), processing (2 to 7 days of age), and weaning (18 to 22 days of age). Additionally, processing fluids and family oral fluids were collected from 2-7 days of age and weaning age, respectively. In farms A and B, PRRSV RNA was detected in tongue tips from all age groups (100 and 95%, respectively). In addition, PRRSV RNA was detected in pooled serum samples (42 and 27%), processing fluids (100 and 50%), and family oral fluids (11 and 22%). Interestingly, the average Ct value from tongue tips was numerically lower than the average Ct value from serum samples in the newborn age. In farm C, PRRSV RNA was only detected in serum samples (60%) and family oral fluids (43%), both from the weaning age. Further, no PRRSV RNA was detected in tongue tips when pooled serum samples from the same age group tested PRRSV RNA-negative. Taken together, these results demonstrate the potential value of tongue tips for PRRSV monitoring and surveillance.

Efficacy of Pooled Serum Internal Quality Control in Comparison with Commercial Internal Quality Control in Clinical Biochemistry Laboratory.

Introduction With increasing automation in clinical laboratories, the requirements for quality control (QC) material have greatly increased in order to monitor performance. The constant use of commercial control material is not economically feasible for many countries because of nonavailability or the high-cost of those materials. Therefore, preparation and use of in-house QC serum will be a very cost-effective measure with respect to laboratory needs. Materials and Methods In-house internal quality control from leftover serum samples of master health checkup subjects, which have been screened negative for HIV, HCV and HBsAg antibodies was pooled in a glass jar with ethanediol as preservative and kept in deep freezer at - 20°C. From the pooled serum, 100 microliter thirty aliquots were prepared. Every day along with commercial internal QC (IQC), one aliquot of pooled serum was analyzed for 30 days for the following parameters: plasma glucose, blood urea, serum creatinine, total cholesterol, triglycerides (TGL), high-density lipoprotein, calcium, total protein, albumin, total bilirubin, AST, ALT, ALP, amylase. After getting 30 values for each parameter, mean, standard deviation (SD) and CV% were calculated for both IQC commercial sample and pooled serum sample. Results The mean, SD, and CV% of glucose, cholesterol, TGL, calcium, alanine aminotransaminase (ALT), aspartate aminotransferase (AST), amylase, and alkaline phosphatase (ALP) were statistically significant between pooled serum and commercial QC. Conclusion In-house QC prepared from pooled serum is better than commercial internal QC. The biochemical parameters were stable in pooled serum due to less matrix effect; also, variation was less in pooled serum IQC.

Investigation of the potential for sera from cattle persistently infected with bovine viral diarrhea virus to generate false-negative antibody ELISA results in pooled serum from seropositive and seronegative cattle.

We investigated the potential for viremic sera from cattle persistently infected with bovine viral diarrhea virus to create false-negative antibody results when testing pools of 10 sera by indirect or blocking ELISAs. Seronegative viremic sera ( n = 23) were each added to a series of artificially constructed pools containing various percentages (0-90%) of antibody-positive sera, and the resulting pools were assayed for antibody. In all 23 cases, a negative antibody result was obtained in the pool containing no seropositive sera. In contrast, all pools containing ≥10% seropositive serum, representing a single seropositive animal in a pool of 10 samples, returned a positive result in both antibody ELISAs. We concluded that the likelihood of a false-negative antibody result occurring as a result of the presence of serum from a viremic animal was low, and therefore did not preclude the use of pooled sera for serosurveillance.

The Antibody Two-Step Solution.

Problems with antibody quality have been described in numerous recent publications.  In the present commentary it is argued that these quality problems are due primarily to issues of antibody variability and antibody validation.  Further it is argued that the problem of antibody variability must be solved before validation can be useful.  A two-step solution to the antibody problem is thus proposed.