Multiplex cytokine profiling with highly pathogenic material: use of formalin solution in luminex analysis

Abstract

Work with highly pathogenic material mandates the use of biological containment facilities, involving microbiological safety cabinets and specialist laboratory engineering structures typified by containment level 3 (CL3) and CL4 laboratories. Consequences of working in high containment are the practical difficulties associated with containing specialist assays and equipment often essential for experimental analyses. In an era of increased interest in biodefence pathogens and emerging diseases, immunological analysis has developed rapidly alongside traditional techniques in virology and molecular biology. For example, in order to maximise the use of small sample volumes, multiplexing has become a more popular and widespread approach to quantify multiple analytes simultaneously, such as cytokines and chemokines. The luminex microsphere system allows for the detection of many cytokines and chemokines in a single sample, but the detection method of using aligned lasers and fluidics means that samples often have to be analysed in low containment facilities. In order to perform cytokine analysis in materials from high containment (CL3 and CL4 laboratories), we have developed an appropriate inactivation methodology after staining steps, which although results in a reduction of median fluorescent intensity, produces statistically comparable outcomes when judged against non-inactivated samples. This methodology thus extends the use of luminex technology for material that contains highly pathogenic biological agents.

Fig. 1

Mean concentrations of 26 cytokines/chemokines in quality control preparations after treatment with 0%, 4% and 10% formalin solution (Error bars denote the standard deviation).

Fig. 2

Effect of 4% and 10% formalin treatment on the MFI values obtained using a preparation of 2000 pg/ml for all of the cytokines/chemokines tested (Error bars denote standard deviation. Untreated samples given a value of 100).

Fig. 3

Effects of 4% and 10% formalin treatment on yield of cytokine in PHA + LPS and PMA + ionomycin stimulated PBMCs from human volunteers (n = 7) compared to untreated samples (Cytokine yield determined by dividing the concentration of formalin-treated samples with untreated sample. Error bars denote standard deviation; Dotted line represents relevant value of untreated sample; Mann–Whitney statistical test: *P < 0.05; **P < 0.01).