Lymphocyte Response Assay: Report on Precision of a Novel Cell Culture Test

Immune tolerance or learned reactivity to the non‐self ‘external’ world is largely epigenetic; acquired immune‐memory carried by a group of specialized white blood cells known as lymphocytes. Activation of lymphocytes, while an important part of normal immune function, is also linked to chronic inflammatory and autoimmune disease. These diseases occur when innate immune‐function overload causes lymphocytes to become activated against ‘self’ tissue. The Lymphocyte Response Assay (LRA) described here (LRA by ELISA/ACT ) addresses the need to distinguish helpful from harmful immune memory responses. Prior LRA methods include colony formation, thymidine incorporation and cytokine release assays. In this LRA, from one ounce of whole blood, 500 separate cell cultures can be performed. In the assay, antigen pre‐coated microtiter plate wells are incubated in autologous cell‐rich plasma under standard physiologic conditions (35±2 °C in a CO 2 and humidity controlled incubator), followed by direct microscopy imaging of lymphocyte‐specific responses. This LRA uses pre‐analytic techniques to avoid activation of blood clotting in the CPDA vacutainer tubes used for specimen collection and transport, with specimen temperature maintained between 4–10°C during shipment. An ex vivo brief cell culture system is followed by a cell‐membrane based, amplified signal, which is transduced into an image that can be graded for clinical interpretation. Positive and negative controls are included with each array. Assay grade is read as: non‐reactivation (<8% reactive cells compared to the internal positive control), moderate reactivation (9–50% reactive cells), and strong reactivation (>50% reactive cells). Reactive cells appear to all be T or B class lymphocytes. From reports currently unpublished, antigen‐presenting cells can be readily observed moving toward lymphocytes prior to cell reactivation. The few hours of incubation used are insufficient for lymphocyte memory to be acquired; thus reactivated cells must have learned to recognize specific antigens prior to phlebotomy. We report based on consecutive blind split samples representing 4138 items tested ( Table 1). Split sample variance was less than 2.3%, qualifying this LRA as a high‐sensitivity (highly reproducible; hsLRA) clinical assay of immune‐tolerance and delayed allergy ( Table 1). Since it is suitable for automation, the assay has the potential for high‐throughput screening, considerably increasing the number and rate of samples which could be analyzed. Finally, the assay needs to be compared with flow cytometry, and in clinical outcome studies, to verify its promising predictive significance as a personalized biomarker. Support or Funding Information Funding from grant to Health Studies Collegium by ELISA/ACT BioTechnologies 1 Performance of high‐sensitivity Lymphocyte Response Assay (hsLRA) on consecutive blind split samples.# items tested # items matched # items non‐matched % of items a matched % of items a non‐matched Test interval (year)636 633 3 99.48±0.91 0.52±0.91 2011 1819 1778 41 97.50±3.58 2.19±3.19 2012 1156 1134 22 98.16±2.11 1.84±2.11 2013 527 505 22 95.56±3.01 3.89±2.69 20144138 4050 88 97.60±3.00 2.25±2.75 Total