Introduction 6 9 5 15 19 5 13 13 3 14 16 8 17 21 22 2 10 1 3 7 10 12 14 20 21 4 18 The present study compares specific circulating lymphocyte subsets in patients with mild psoriasis, patients with moderate-to-severe psoriasis and normal subjects. Materials and methods Patients and controls 4 18 Patients were free of any systemic therapy for at least 4 weeks and did not use any topical therapy in the last 2 weeks. Peripheral blood was obtained from all subjects with their written informed consent. Control samples were collected from 10 healthy volunteers without any history or signs of skin disease (four male and six female aged 24–49, mean age 33.8 years). Preparation of PBMC’s For each patient, the exact amount of blood withdrawn was determined by measuring the height of the column of blood in the tube, which was subsequently converted to the corresponding volume in microliter. PBMC’s were isolated from heparinized blood by density centrifugation on polyester gel (Becton Dickinson Vacutainer™ CPT™, Franklin Lakes NJ, USA). After filtering through a 70 μm cellstrainer, cells were washed twice. 5 −1 The following monoclonal antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-CD4, FITC-conjugated anti-CD8, phycoerythrin (PE)-conjugated anti-CD4, PE-conjugated anti-CD8, PE-conjugated anti-CD94 and PE-conjugated anti-CD161 (all from Immunotech, Marseille, France), as well as FITC-conjugated anti-CD45RA (Becton Dickinson, San Jose, CA, USA) and FITC-conjugated anti-CD45RO (DAKO, Glostrup, Danmark). Flowcytometric analysis Cells were analyzed with an EPICS Elite flow cytometer (Coulter, Luton, UK), using the forward scatter as a discriminator. Lymphocytes were identified by gating on CD45 and side and forward scatter properties. All samples were processed within 18 h of phlebotomy. The following (combinations of) PE and FITC-conjugated reagents were used to determine the expression of each antigen or antigen combination on lymphocytes derived from peripheral blood: CD4+ (marker for T-helper cells), CD8+ (marker for cytotoxic T cells), CD45RA+ (marker for naïve T-cells), CD45RO+ (marker for memory T-cells), CD94+ (marker for NK-cells, T cells), CD161+ (marker for NK-cells, T-cells with memory phenotype), CD4+ CD45RA+, CD4+ CD45RO+, CD8+ CD45RA+, CD8+ CD45RO+, CD8+ CD94+ and CD8+CD161+. Determination of absolute numbers of lymphocytes −1 Ratios for CD4+/CD8+ cells, CD45RO+/CD45RA+ cells, CD4+CD45RO+/CD4+CD45RA+ cells and CD8+CD45RO+/CD8+CD45RA+ cells were calculated afterwards, with data derived from this analysis. Statistical analysis p Results 1 Table 1 Demographic and psoriasis related characteristics of patient and subjects participating in the study (mean ± SEM)   Moderate-to-severe psoriasis Mild psoriasis Normal subjects Demographics Age (years) 46.2 ± 3.45 52.8 ± 3.49 33.8 ± 3.1 Male:female ratio  12:3 7:5 4:6 Psoriasis related characteristics PASI-score  20.97 ± 2.55 6.11 ± 1.27 NA Duration (years) 16.7 ± 2.7 22.2 ± 2.5 NA Total cell counts −1 −1 −1 p The total number of lymphocytes, and the CD4+ and CD8+ counts of the patients with moderate-to-severe disease were also routinely measured at the GLP certified Central Haematology Laboratory in our hospital, in which the whole blood lysing method is the standard. Comparison of both methods reveals an acceptable resemblance in results, with lower counts of maximum 10%. Lymphocyte subsets 1 p Fig. 1 p  No significant differences were observed between patients with mild psoriasis and normal subjects. p 2 3 Fig. 2 p  Fig. 3 p  Lymphocytes expressing NK cell receptors p p 4 Fig. 4 p p  p p 5 Fig. 5 p  Correlation between PASI-score and lymphocyte subsets and cells expressing NK receptors Linear regression analysis revealed no significant correlation between the individual PASI-scores and the quantifications of lymphocyte subsets or cells with NK-cell receptors. Correlation analysis between disease activity and peripheral lymphocytes did not reveal a difference between patients with spreading lesions and those with stable plaque psoriasis. Discussion 4 18 10 3 10 10 11 p 22 22 3