Non–heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel

Abstract

Background: Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets. Objective: To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT. Methods: Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1β) in the absence or presence of NiSO 4 . The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSE low/neg CD3 + CD4 + T-lymphocytes upon nickel stimulation compared to the percentage of CFSE low/neg CD3 + CD4 + T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test.Results: Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4 + cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails. | 723 de GRAAF et Al.

Memory T helper cells identify patients with nickel, cobalt, and chromium metal allergy

“…In agreement with this study, Spoerri et al 15 reported comparable sensitivity and specificity (75% vs 80%), respectively, of Ni‐specific Th cells as seen in this study (69% and 85%), using the same culture conditions and flow cytometric LPT analysis. By contrast, de Graaf et al 14 found a low sensitivity of 35% and high sensitivity of 93% of Ni‐specific Th cells, when culturing the cells in a medium with 10% heat‐inactivated serum. Furthermore, de Graaf et al 14 reported that in contrast to stimulation with various cytokine cocktails, nonheated inactivated autologous serum albumin resulted in 95% sensitivity and 93% specificity.…”

“…By contrast, de Graaf et al 14 found a low sensitivity of 35% and high sensitivity of 93% of Ni‐specific Th cells, when culturing the cells in a medium with 10% heat‐inactivated serum. Furthermore, de Graaf et al 14 reported that in contrast to stimulation with various cytokine cocktails, nonheated inactivated autologous serum albumin resulted in 95% sensitivity and 93% specificity. The nonheated inactivated autologous serum is indeed an attractive approach to optimizing the LPT; however, the influencing factors in the allergic individuals' autologous serum are still unknown, making, for example, fluctuations of these factors a potential problem.…”

In Vitro Monitoring of Human T Cell Responses to Skin Sensitizing Chemicals—A Systematic Review

“…Conditions for T cell activation may be optimized. Besides, depletion of regulatory T cells (e.g., CD25+ T cells), addition of cytokines such as IL-12 or IL-4 or autologous serum may support the proliferation of chemical-specific T cell subsets [ 95 , 127 , 134 , 135 ]. In addition, the presence of co-stimulatory antibodies (e.g., α-CD28, α-CD49a) or checkpoint inhibitor antibodies (e.g., α-programmed death ligand 1/2 (PD-L1/2), α-PD-1, α-cytotoxic T-lymphocyte-associated protein (CTLA)-4) may optimize conditions for T cell activation [ 136 ].…”

Patch test–relevant concentrations of metal salts cause localized cytotoxicity, including apoptosis, in skin ex vivo

“…The challenge will be to remain above the elicitation threshold at noncytotoxic metal concentrations. Alternatively, focusing on in vitro lymphocyte cytokine and transformation tests which are showing promising results for identifying people with allergy to metals is an option 55,56 …”