Comparing two ways of mixing PBMCs and washing medium by viability (A) and absolute count of live PBMCs (B).

<p>Paired samples 20 donors included. Ref.: reference group. NS: non-significant. **: p<0.01.</p

PBMC viability (A) and absolute live PBMC recovery (B) by incubation.

<p>Samples were stored in a 37°C incubator with 5% CO₂. Paired samples from 20 donors were included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

Impact of washing-medium temperature on viability (A) and PBMC recovery (B).

<p>20 donors included. Ref.: reference group. **: p<0.01.</p

Centrifugation time and force and PBMC viability (A) and absolute live PBMC count (B).

<p>Paired samples from 18 donors included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

PBMC viability (A) and absolute recovery (B) as a result of varying thawing time in 37°C heated water bath.

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p

Temperature of centrifuge by PBMC viability (A) and absolute live PBMC recovery (B).

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p

Gating strategy to identify live/dead CD45+ leukocytes.

<p>A) Events were triggered on FSC-H at a deliberately low threshold to avoid accidental exclusion of dead cells. B) Doublet exclusion C) Identification of CD45 positive cells D) Dead cells identified as 7-AAD positive events. Compensation for spectral overlap was not required.</p

Additional file 2: of Use of prescription drugs and risk of postoperative red blood cell transfusion in breast cancer patients: a Danish population-based cohort study

Supplementary Figure S1 and Supplementary Tables S1–S5. Figure S1. Flow diagram. Table S1. Specific comorbid conditions included in the Charlson Comorbidity Index, according to use of selected prescription drugs. Table S2. Risk and crude and adjusted odds ratios for blood transfusion within 7 days of surgery among 22,238 breast cancer patients, according to use of selected prescription drugs. Table S3. Risk and crude and adjusted odds ratios for postoperative blood transfusion within 14 days of surgery among 22,238 breast cancer patients, according to use of selected prescription drugs and with the exposure window defined as 1–30 days before surgery. Table S4. Risk and crude and adjusted odds ratios for postoperative blood transfusion within 14 days of surgery among 22,238 breast cancer patients, according to use of selected prescription drugs and adjusted for selected comorbidities (cardiac disease, chronic pulmonary disease, and diabetes). Table S5. Risk and crude and adjusted odds ratios for postoperative blood transfusion within 14 days of surgery among 21,578 breast cancer patients according to use of selected prescription drugs, with the exposure window defined as 1–30 days before surgery and after excluding patients with anemia [< 12 g/dL (7.4 mmol/L)]. (ZIP 346 kb

The Impact of B-Cell Perturbations on Pneumococcal Conjugate Vaccine Response in HIV-Infected Adults

<div><p>Untreated HIV infection results in severe perturbations of the B-cell population and hyporesponsiveness to vaccination. We studied associations between circulating B-cell subsets and antibody response to pneumococcal conjugate vaccine in treated and untreated HIV patients.</p> <p>Ninety-five HIV-infected adults were grouped according to antiretroviral therapy (ART) and CD4+ cell count as follows: 20 ART-naïve (no prior ART), 62 ART-responders (received ART, and CD4 count >500 cells/µl), and 13 impaired responders (received ART for more than 3 years, and CD4 count <500 cells/µl). All subjects were immunized twice with double-dose 7-valent pneumococcal conjugate vaccine with or without 1 mg CPG 7909 (toll-like receptor 9 agonist) at baseline and after three months. Pre-vaccination B-cell subpopulations were assessed by flow cytometry. Serum IgG concentrations for vaccine serotypes were quantified by ELISA at baseline and 3, 4, and 9 months post-vaccination. ART responders had more isotype-switched memory B cells and more marginal-zone (MZ)-like B cells compared with impaired responders. Furthermore, ART-naïve patients had higher concentration of transitional B cells and plasmablasts compared with B cells of other patient groups. The concentration of MZ-like, isotype switched memory cells and plasmablasts correlated positively with post-vaccination IgG concentration at 3, 4, and 9 months. Low concentrations of isotype-switched memory B cells was the strongest independent predictor of poor pneumococcal conjugate vaccine responsiveness, emphasizing that B-cell subset disturbances are associated with poor vaccine response among HIV-infected patients</p> </div

Adjusted regression analysis: Baseline B-cell subpopulations as predictors for IgG antibody titers.

<p>IgG antibody concentration is calculated as the relative vaccine-specific IgG increase of the 7 <i>Streptococcus pneumoniae</i> polysaccharide serotypes contained in the PCV-7 vaccine from pre-vaccination baseline to 3, 4 and 9 months, measured by ELISA.</p><p>B-cell counts are calculated as the routine lab lymphocyte count multiplied with the B-cell percentage of lymphocytes measured by flow cytometry.</p><p>Adjusted for patient group (‘ART-naïve’, ‘impaired responders’, ‘ART-responders’), CPG7909 adjuvant (yes/no) and current smoker (yes/no).</p><p>RC; Regression Coefficient (increase in log(µg IgG/ml) with 10-fold increase in cell counts/fractions), p; p-value, n; number of participants.</p><p>Transitional; IgD+, IgM+, CD27−, CD38+ B cells, Naive; IgD+, IgM+/−, CD27−, CD38− B cells, MZ-like; marginal zone-like B cells (IgD+, IgM+, CD27+, CD38−), IgM-only memory; IgD−, IgM+, CD27+, CD38− B cells, ITS memory; isotype switched memory B cells (IgD−, IgM−, CD27+, CD38−), ITS plasmablasts; isotype switched plasmablasts (IgD−, IgM−, CD27+, CD38+).</p