A comparison of manual counting of rabbit reticulocytes with ADVIA 2120i analyzer counting

We compared manual counting of reticulocytes in rabbits with automatic counting using an ADVIA 2120i analyzer. Reproducibility and the influence of different anticoagulants (EDTA and Li-heparin) were also examined. Blood samples of 331 rabbits (method comparison, n = 289; reproducibility, n = 33; comparison of anticoagulants, n = 9) were tested. The reticulocyte numbers of each specimen were manually determined twice for method comparison. Passing–Bablok regressions, Bland–Altman plots, and the coefficient of variation (CV) were used to evaluate statistical significance. Good correlation (rs = 0.81) was observed between manual reticulocyte counting (groups 1–4) and the ADVIA 2120i. Quantification with the ADVIA 2120i was reproducible for relative reticulocyte numbers (EDTA, CV = 4.24%; Li-heparin, CV = 3.63%) and absolute reticulocyte numbers (EDTA, CV = 5.64%; Li-heparin, CV = 3.81%). The absolute and relative reticulocyte numbers were significantly higher in Li-heparin samples than in EDTA samples (absolute, p = 0.009; relative, p = 0.016). The ADVIA 2120i is suitable for counting reticulocytes in rabbit blood samples, but reticulocyte numbers are higher by manual counting than by ADVIA 2120i counting. Therefore, microscopic confirmation of quantifications is recommended when high numbers of reticulocytes are observed. The anticoagulant of choice is EDTA

Method Validation and Establishment of Reference Intervals for an Insulin-like Growth Factor-1 Chemiluminescent Immunoassay in Cats

Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats

Cutting Edge: Human γδ T Cells Are Activated by Intermediates of the 2- C

Activation of V gamma 9/V delta 2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C:-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the V gamma 9/V delta 2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate gamma delta T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome