Flow cytometry is an important technique for characterization of immune cells, with accurate lymphocyte profiling being essential for clinical diagnostics and research applications. While immediate processing of blood samples is ideal, long-term storage solutions are needed for large-scale studies or settings without immediate access to laboratory facilities. TransFix® is a chemical stabilization solution that allows delayed analysis by preserving cell morphology and surface markers. However, the impact of long-term cryopreservation in TransFix® on lymphocyte integrity remains underexplored. In this study, we evaluated the efficacy of cryopreservation in TransFix® for maintaining the proportions of key lymphocyte subsets, including CD3⁺ T cells, CD3⁺CD4⁺ T helper cells, CD3⁺CD8⁺ cytotoxic T cells, CD19⁺ B cells, and CD16⁺/CD56⁺ natural killer cells. Blood samples were cryopreserved in TransFix® for varying time periods, up to 48 months, and compared to fresh samples using flow cytometry. The results show that the proportions of lymphocyte subsets remain stable during cryopreservation for up to 48 months, with no significant differences observed between fresh and cryopreserved samples. This suggests that TransFix® can successfully preserve lymphocyte integrity for long-term storage, providing a reliable option for delayed analysis. These results highlight the usefulness of TransFix® for studies that require extended storage, making it easier to conduct immune monitoring in a wide range of settings.

Autoimmune thyroid disease (AITD) is associated with other autoimmune endocrine diseases such as type 1 diabetes (T1D) and celiac disease (CeD). Thyroid peroxidase autoantibodies (TPOA) are biomarkers of AITD but may also occur in patients with other autoimmune diseases. We examined cross-sectional correlations between TPOA and an array of immune markers in a cohort of 90 children with exclusively T1D (n = 27), CeD (n = 16) or a combination of these two diseases (n = 18), compared to a reference group of children without these diagnoses (n = 29). Children with exclusively T1D or T1D in combination with CeD had higher levels of TPOA with an overrepresentation among girls. The correlations between immune markers and TPOA were distinctly different between all study groups. In children with T1D, TPOA correlated mainly with the T helper 1 associated IFN-γ and pro-inflammatory IL-1β. In contrast, in children with combined diagnoses, TPOA was correlated with pro-inflammatory MCP-1, the acute phase proteins ferritin, fibrinogen, and serum albumin A, and adipocytokines resistin and visfatin. Children with exclusively CeD did not show the same strong association between immune markers and TPOA. In conclusion, TPOA positivity was mainly detected in patients with T1D and female sex. Several inflammatory markers correlated with TPOA, indicating a relation to autoimmune parameters in children with T1D, CeD or both, but preceding symptoms AITD.

Immunotherapies aimed at preserving residual beta cell function in type 1 diabetes have been successful, although the effect has been limited, or raised safety concerns. Transient effects often observed may necessitate redosing to prolong the effect, although this is not always feasible or safe. Treatment with intralymphatic GAD-alum has been shown to be tolerable and safe in persons with type 1 diabetes and has shown significant efficacy to preserve C-peptide with associated clinical benefit in individuals with the human leukocyte antigen DR3DQ2 haplotype. To further explore the feasibility and advantages of redosing with intralymphatic GAD-alum, six participants who had previously received active treatment with intralymphatic GAD-alum and carried HLA DR3-DQ2 received one additional intralymphatic dose of 4 μg GAD-alum in the pilot trial DIAGNODE-B. The participants also received 2000 U/day vitamin D (Calciferol) supplementation for two months, starting one month prior to the GAD-alum injection. During the 12-month follow-up, residual beta cell function was estimated with Mixed-Meal Tolerance Tests, and clinical and immune responses were observed. C-peptide decreased minimally, and most patients showed stable HbA1c and IDAA1c. The mean % TIR increased while the mean daily insulin dose decreased at month 12 compared to the baseline. Redosing with GAD-alum seems to be safe and tolerable, and may prolong the disease modification elicited by the original GAD-alum treatment.

Background/Objectives: Flow cytometry is a convenient tool in immunophenotyping for monitoring the status of immunological conditions and diseases. The aim of this study was to investigate the effect of isolation and cryopreservation by flow cytometric analysis on subpopulations of CD4+ T helper (Th), T regulatory (Treg), CD8+ T cytotoxic (Tc), CD56+ NK, CD19+ B and monocytes. Freshly isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) were compared to fresh whole blood.

Methods: Peripheral blood was collected from healthy donors and prepared for flow cytometric analysis using the same panels of antibodies throughout the study.

Results: Comparisons between fresh (F)- and cryopreserved (C)-PBMCs showed no major differences in percentages of CD4+, Th1, Th2 and CD4+CD25+CD127low Treg cells. No differences in percentage of CD8+ or subpopulations of naive/stem, central or effector memory cells were observed between F- and C-PBMCs. The percentage of CD56+ NK cells, CD19+ B cells or classical and nonclassical monocytes did not differ between F-and C-PBMCs either. On the contrary, whole blood had lower percentages of Th and NK cells but higher percentages of Th1, Th17, Th1Th17, Tregs, Tc and B cells compared to C-PBMCs, while it had a higher proportion of Tc compared to F-PBMCs.

Conclusions: Flow cytometric immunophenotyping minimally differs between freshly isolated and cryopreserved PBMCs. This implies the possibility of cryostorage of cohorts for later analysis. Importantly, care must be taken when comparing results from whole blood with isolated and cryopreserved PBMCs. Collectively, these results can contribute to the standardization of flow cytometric protocols in both clinical and research settings.

Type 1 diabetes (T1D) and celiac disease (CeD) are common autoimmune diseases in children where the pathophysiology is not fully characterized. The autoimmune process involves a complex scenario of both inflammatory and regulatory features. Galectin-1 (GAL-1) has a wide range of biological activities e.g., interaction with immune cells. We examined the relationship between GAL-1 and soluble immune markers and T cell subsets in a cohort of children with T1D and/or CeD relative to healthy children. Galectin-1, together with several soluble immune markers (e g interleukins (IL)), tumor necrosis factor (TNF), acute phase proteins and matrix metalloproteinases (MMP) were measured in sera from children with T1D and/or CeD by fluorochrome (Luminex) technique using children without these diseases as a reference. Subgroups of T cells, including T regulatory (Treg) cells, were analyzed by flow cytometry. Association between GAL-1, pro-inflammatory markers and Treg cells differed depending on which illness combination was present. In children with both T1D and CeD, GAL-1 correlated positively with pro-inflammatory markers (IL-1β, -6 and TNF-α). Composite scores increased the strength of correlation between GAL-1 and pro-inflammatory markers, Th1-associated interferon (IFN)-γ, and T1D-associated visfatin. Contrary, in children diagnosed with exclusively T1D, GAL-1 was positively correlated to CD25hi and CD25hiCD101+ Treg cells. For children with only CeD, no association between GAL-1 and other immune markers was observed. In conclusion, the association observed between GAL-1, soluble immune markers and Treg cells may indicate a role for GAL-1 in the pathophysiology of T1D and, to some extent, also in CeD.

Our purpose was to characterize the pattern of B-cell subsets in children with a combined diagnosis of type 1 diabetes (T1D) and celiac disease (C) since children with single or double diagnosis of these autoimmune diseases may differ in peripheral B-cell subset phenotype patterns.

B-cells were analyzed with flow cytometry for the expression of differentiation/maturation markers to identify transitional, naive and memory B-cells. Transitional (CD24hiCD38hiCD19+) and memory Bregs (CD24hiCD27+CD19+, CD1d+CD27+CD19+, CD5+CD1d+CD19+) were classified as B-cells with regulatory capacity.

Children with a combined diagnosis of T1D and C showed a pattern of diminished peripheral B-cell subsets. The B-cells compartment in children with combined diagnosis had higher percentages of memory B subsets and Bregs, including activated subsets, compared to children with either T1D or C. Children with combined diagnosis had a lower percentage of naive B-cells (CD27-CD19+; IgD+CD19+) and an increased percentage of memory B-cells (CD27+CD19+; IgD-CD19+). A similar alteration was seen among the CD39+ expressing naive and memory B cells. Memory Bregs (CD1d+CD27+CD19+) were more frequent, contrary to the lower percentage of CD5+ transitional Bregs in children with a combined diagnosis. In children with either T1D or C, the peripheral B-cell compartment was dominated by naive cells.

Differences in the pattern of heterogenous peripheral B-cell repertoire subsets reflect a shifting in the B-cell compartment between children with T1D and/or C. This is an immunological challenge of impact for the pathophysiology of these autoimmune diseases.

Type 1 diabetes (T1D) and celiac disease are both characterized by an autoimmune feature. The diseases also share the same risk genes, and thereby patients have an increased risk of developing the other disease subsequently. The pattern of peripheral T and B cell subsets and soluble immune markers (cytokines, chemokines, acute phase proteins, adipocytokines and matrix metalloproteinases) are not yet well characterized in children with a combination of the common pediatric immunological disorders, T1D and celiac disease. To better understand the complex pathophysiology, it is important to gain a deeper knowledge of alterations present in the peripheral immune profile in children with these autoimmune diseases. Pinpointing biomarkers, e.g. peripheral immune markers, can hopefully contribute to the improvement of prognosis, diagnosis, and disease management. Flow cytometry is useful for studying different immune cells, but several pre-analytical factors may affect the outcome. In order to generate reliable results, it is important to evaluate the impact of different pre-analytical factors that possibly can lead to in vitro alterations of the immune cells.

Aim: The overall aim of this thesis was to increase our knowledge of peripheral immune marker patterns in children with a combined diagnosis of T1D and celiac disease, with a focus on T and B cell subsets and soluble immune markers; by immunological methods evaluated and adapted for this purpose.

Methods: This thesis comprises methodological and cross-sectional studies. The methodological studies are based on whole blood collected from sixty blood donors to examine the impact of pre-analytical factors (anticoagulant, sample handling time, isolation and cryopreservation) that may affect the immune cells (Study I, II). The cross-sectional studies include blood samples collected from a total of 103 participants (children with T1D and/or celiac disease or no diagnosis at all). The pattern of peripheral B (Study II) and T (Study III) cell subsets were examined by flow cytometry. Nearly thirty soluble immune markers were quantified in serum by Luminex technology (Study IV).

Results: Peripheral lymphocytes were stable in whole blood samples up to 24 hours, regardless of the anticoagulant. Generally, T and B cell subsets were not affected by isolation and cryopreservation. Children with combined T1D and celiac disease had higher percentages of terminally differentiated memory T helper cells, lower percentages of effector memory T cytotoxic cells and lower expression of suppressive immune markers on regulatory T cells compared with the other study groups. Further, children with combined T1D and celiac disease had a higher percentage of memory B and lower percentages of naive B cells than children with either T1D or celiac disease. Contrary, children with single diagnoses had an inverted naive/memory B cell pattern compared to children with combined diagnoses. Several of the "classical" (cytokines, chemokines), as well as "non-classical" (acute phase proteins, adipocytokines, matrix metalloproteinases) immune markers, were lower in children with combined diagnoses compared to the other study groups.

Conclusions: Based on our results, we conclude that whole blood samples stored up to 24 hours are feasible for flow cytometric analysis of lymphocyte subsets, regardless of the type of anticoagulant. Further, isolated and cryopreserved immune cells are feasible for flow cytometric analysis of T and B cell subsets. Impairment in the T and B cells mediated immune regulation in children with combined T1D and celiac disease seems to be clearly divergent from those seen in children with exclusively one of these two autoimmune diseases. Children with combined T1D and celiac disease appear to have a suppressed immune profile, including "classical" and "non-classical" immune markers. The methodological studies provide deeper knowledge of how reliable results can be obtained in studies of peripheral immune cells, e.g., in children with autoimmune diseases. The knowledge obtained by this thesis also brings a better understanding of the pattern of peripheral immune markers in T1D and/or celiac disease. This could potentially contribute to promoting the improvement of prognosis, diagnosis, and disease management.

Children diagnosed with a combination of type 1 diabetes (T1D) and celiac disease (CD) show a dysregulated T helper (Th) 1/Th17 response. Besides the cellular involvement, several soluble immune markers are involved in the autoimmune process of both T1D and CD. Only few studies have examined the peripheral pattern of different cytokines, chemokines, and acute phase proteins (APP) in children with combined T1D and CD. To our knowledge no studies have evaluated the serum levels of adipocytokines and matrix metalloproteinases (MMPs) in this context. The purpose of the present study was to acquire more knowledge and to gain deeper understanding on peripheral immunoregulatory milieu in children with both T1D and CD. The study included children diagnosed with both T1D and CD (n=18), children with T1D (n=27) or CD (n=16), and reference children (n=42). Sera were collected and analysis of twenty-eight immune markers (cytokines, chemokines, APPs, adipocytokines and MMPs) was performed with Luminex technique. The major findings showed that children with double diagnosis had lower serum levels of interleukin (IL)-22, monocyte chemoattractant protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, procalcitonin, fibrinogen, visfatin and matrix metalloproteinase (MMP)-2. These results indicate a suppressed immune profile in children with combined T1D and CD, including Th17 cytokines, chemokines, acute phase proteins, adipocytokines and MMPs. We conclude that besides cytokines and chemokines, other immune markers, e.g. APPs, adipocytokines and MMPs, are of importance for further investigations to elucidate the heterogeneous immune processes present in patients diagnosed with T1D in combination with CD.

Introduction: Cytokines, chemokines, acute phase proteins (APP), adipocytokines and matrix metalloproteinases (MMP) are involved in different pathophysiological processes of inflammatory character. The role of the different immune markers and the peripheral immunoregulatory milieu in children diagnosed with type 1 diabetes (T1D) in combination with celiac disease (CD) is not fully understood and is not well studied. The purpose of the present study was therefore to acquire more knowledge and to gain deeper understanding on peripheral immunoregulatory milieu in children with T1D and/or CD.

Methods: The study included children diagnosed with T1D in combination with CD (n=18), children with T1D (n=27) or CD (n=16), and reference children (n=42).

Blood samples were collected, and serum stored in -80°C until analysis, avoiding multiple freeze-thaw cycles. The inflammatory cyto/chemokines (IL-1β, -5, -6, -8, -9, -10, -13, -15, -17A, -22, -25, -33, IFN-γ, TNF-α, G-CSF, MCP-1, MIP-1α, MIP-1β), diabetes related immune markers (visfatin, resistin), APP (procalcitonin (PTC), ferritin, tissue protein activator, fibrinogen, serum amyloid A) and matrix metalloproteinases (MMP-1, -2, -3) were analyzed with Luminex technique using Bio-Plex assays. Hierarchical cluster analysis was used to identify similarities/differences in immune profiles between children with double diagnosis and children with single diagnosis and reference children. Mann-Whitney U test was used for comparison of the different diagnosis groups within the clusters and whole cohort, respectively.

Results: The largest cluster included 75% of the participants and the diagnose distribution in the cluster were very similar to the distribution in the whole study cohort. The remaining 25% were divided in two smaller clusters representing 15.5% and 6.5% respectively. The major finding of this study showed that children with double diagnosis had (1) lower serum levels of IL-22, MCP-1, PCT, visfatin and MMP-2 compared to children with T1D; and (2) lower serum levels of the APC associated chemokine MIP-1α compared to reference children, observed in the main cluster. Most of these observations were also seen in the whole cohort.  

Conclusion: Our observations indicate decreased serum levels of IL-22, MIP-1α, MCP-1, PCT, visfatin and MMP-2 in children diagnosed with T1D in combination with CD. These results indicate a suppressed immune profile including Th17 cytokines, chemokines, acute phase proteins, diabetes-related and matrix metalloproteinase immune markers. Functional studies of the involved immune cells (CD4⁺ Treg, CD8⁺ Treg, NK-cells and dendritic cells) could contribute to elucidate the heterogeneous immunological processes in children with more than one autoimmune disease.