Whole blood cytokine release assays reveal disparity between capillary blood sampling methods.

INTRODUCTION: The use of whole blood in rapid cytokine release assays (CRAs) is becoming an established technique for screening immune responses following natural infection or vaccination, especially in the context of the SARS-CoV-2 pandemic. Establishing an accurate capillary blood sampling method to replace the need for venipuncture could make CRAs more accessible. In this study, capillary blood was collected via two different methods alongside traditional venipuncture to investigate whether the method of blood draw affects cytokine quantification when performing CRAs. METHODS: Adults previously vaccinated with SARS-CoV-2 vaccines donated three blood samples: one by venipuncture, one by finger prick, and one by a microneedle device. Whole blood was aliquoted and incubated overnight with SARS-CoV-2 peptides or left unstimulated. Cytokine release in plasma was measured by multiplex array. RESULTS: In unstimulated samples, little to no cytokines were detected in blood collected via venipuncture or by microneedle devices. Conversely, capillary blood collected by finger prick showed detectable levels of all cytokines analysed, with significantly inflated levels of TNFα, IL-10 (p < 0.0001), IL-2, GM-CSF, and IL-13 (p < 0.01), and 53% of these samples were also positive for IFN-γ. Following peptide stimulation, 25% of samples collected via finger prick showed dysregulated production of IFN-γ, TNFα, IL-2, and IL-10, with lower cytokine production than unstimulated controls. Contrastingly, this was seen in just 4% of venous blood samples and in none of the microneedle samples. CONCLUSIONS: Capillary blood draw via a microneedle device results in highly comparable immune responses to those seen via venipuncture at baseline and following peptide stimulation, suggesting this is a viable method for rapid whole blood CRAs. Conversely, differential cytokine production is observed following capillary blood draw via finger prick.

Differential cellular and humoral immune responses in immunocompromised individuals following multiple SARS-CoV-2 vaccinations.

Introduction: The heterogeneity of the immunocompromised population means some individuals may exhibit variable, weak or reduced vaccine-induced immune responses, leaving them poorly protected from COVID-19 disease despite receiving multiple SARS-CoV-2 vaccinations. There is conflicting data on the immunogenicity elicited by multiple vaccinations in immunocompromised groups. The aim of this study was to measure both humoral and cellular vaccine-induced immunity in several immunocompromised cohorts and to compare them to immunocompetent controls. Methods: Cytokine release in peptide-stimulated whole blood, and neutralising antibody and baseline SARS-CoV-2 spike-specific IgG levels in plasma were measured in rheumatology patients (n=29), renal transplant recipients (n=46), people living with HIV (PLWH) (n=27) and immunocompetent participants (n=64) post third or fourth vaccination from just one blood sample. Cytokines were measured by ELISA and multiplex array. Neutralising antibody levels in plasma were determined by a 50% neutralising antibody titre assay and SARS-CoV-2 spike specific IgG levels were quantified by ELISA. Results: In infection negative donors, IFN-γ, IL-2 and neutralising antibody levels were significantly reduced in rheumatology patients (p=0.0014, p=0.0415, p=0.0319, respectively) and renal transplant recipients (p<0.0001, p=0.0005, p<0.0001, respectively) compared to immunocompetent controls, with IgG antibody responses similarly affected. Conversely, cellular and humoral immune responses were not impaired in PLWH, or between individuals from all groups with previous SARS-CoV-2 infections. Discussion: These results suggest that specific subgroups within immunocompromised cohorts could benefit from distinct, personalised immunisation or treatment strategies. Identification of vaccine non-responders could be critical to protect those most at risk.

Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood.

T cell immunity to natural SARS-CoV-2 infection may be more robust and longer lived than Ab responses. Accurate assessment of T cell responses is critical for understanding the magnitude and longevity of immunity across patient cohorts, and against emerging variants. By establishing a simple, accurate, and rapid whole blood test, natural and vaccine-induced SARS-CoV-2 immunity was determined. Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with previous PCR-confirmed infection, suspected infection, or with no exposure history (n = 128), as well as in donors before and after vaccination (n = 32). Longitudinal assessment of T cell responses following initial vaccination and booster vaccination was also conducted (n = 50 and n = 62, respectively). Cytokines were measured by ELISA and multiplex array. IL-2 and IFN-γ were highly elevated in PCR-confirmed donors compared with history-negative controls, with median levels ∼33-fold and ∼48-fold higher, respectively. Receiver operating curves showed IL-2 as the superior biomarker (area under the curve = 0.9950). Following vaccination, all donors demonstrated a positive IL-2 response. Median IL-2 levels increased ∼32-fold from prevaccination to postvaccination in uninfected individuals. Longitudinal assessment revealed that T cell responses were stable up to 6 mo postvaccination. No significant differences in cytokine production were observed between stimulations with Wuhan, Delta, or Omicron peptides. This rapid, whole blood-based test can be used to make comparable longitudinal assessments of vaccine-induced T cell immunity across multiple cohorts and against variants of concern, thus aiding decisions on public health policies.