A multicolor flow cytometric assay for measurement of platelet-derived microparticles.

INTRODUCTION: Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for "bedside" analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements. MATERIALS AND METHODS: PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments. RESULTS: Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100-700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were <10%, and the variation between two cytometers in two different laboratories was <5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls (p<0.001). CONCLUSIONS: The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples.

CD34+ cell subpopulations detected by 8-color flow cytometry in bone marrow and in peripheral blood stem cell collections: application for MRD detection in leukemia patients.

Fast development in polychromatic flow cytometry (PFC) makes it possible to study CD34+ cells with two scatter and eight fluorescence parameters. Minimal residual disease (MRD) is determined as persistence of leukemic cells at submicroscopic levels in bone marrow (BM) of patients in complete remission. MRD can be present in collections of hematopoietic stem cell from blood (HSC-B). Using PFC, we have defined patterns of antigen expression in CD34+ cell subpopulations in BM and applied them as templates in MRD analysis. Twelve BM samples from hospital control (HC) patients with no signs of hematological malignancy were studied using five 8-color monoclonal antibody combinations detecting subsets of CD34+ cells. These patterns have been used as templates to determine levels of MRD in HSC-B collections from six AML patients. Several subsets of CD34+ precursor cells were found to be present at very low frequencies (<10(-4)) in BM and/or HSC-B collections. All six HSC-B collections from AML patients showed MRD by 8-color technique and only three by previously applied 3-color method. The 8-color technique showed promising results in efficient detection of different CD34+ subpopulations of HSC-B and in MRD quantification. Monitoring of MRD should become a part of quality control of HSC-B collections.

Effects of intercept pathogen inactivation on platelet function as analysed by free oscillation rheometry.

INTRODUCTION: The Intercept Blood System, using InterSol as additive solution, is used for inactivation of contaminating pathogens in PCs, thus reducing the risk for transfusion transmitted infection and making it possible to prolong the storage period. This study aimed at investigating the ability of Intercept treated platelets to induce clot formation, as measured by coagulation time using free oscillation rheometry (FOR), and to compare with that of platelets in concentrates with the additive solution T-Sol or plasma. METHODS: Seventy-four single-donor platelet units were diluted in InterSol (n=27) or T-Sol (n=47) to a mean plasma concentration of 38%. The Intercept treatment was performed by addition of amotosalen HCl to the InterSol PCs followed by UVA irradiation and treatment with a compound adsorption device (CAD). Forty-six units were collected and stored in 100% plasma for comparison. Clotting time was measured by FOR in fresh PCs (within 26h after collection) after stimulation by a platelet activator. Soluble P-selectin was analysed as a marker of platelet activation in the Intercept and T-Sol PCs. RESULTS: The clotting time was shorter for Intercept treated platelets compared to platelets in T-Sol and plasma (p<0.05). There was no difference in clotting time between T-Sol and plasma PCs. Soluble P-selectin was higher for Intercept platelets than platelets in T-Sol (p<0.05). CONCLUSIONS: The platelets treated with the Intercept procedure had good clot promoting capacity.

Monocyte enriched apheresis for preparation of dendritic cells (DC) to be used in cellular therapy.

We describe that with one leukapheresis procedure it is feasible to obtain sufficient numbers of monocytes to be utilized in dendritic cell therapies. Twenty-two leukaphereses were performed on eight healthy volunteers and 13 cancer patients, using Cobe Spectra. An on-line sample was drawn as soon as a stable interface was established. The concentration of monocytes in the sample was used to calculate the volume to be collected to reach target numbers of monocytes. A recovery unit was used to calculate the efficacy of the leukaphereses and we demonstrate an efficacy for monocytes correlating with the amount of processed blood.

Stimulation of T-cell cytokine production and NK-cell function by IL-2, IFN-alpha and histamine treatment during remission of non-Hodgkin's lymphoma.

Limited therapeutic options remain for patients with relapsing lymphoma following chemotherapy and autologous stem cell transplantation (ASCT), hence motivating investigations of complementary treatments. The aim of the present study was to evaluate feasibility and immunological effects of an immunotherapy schedule administered during chemotherapy-induced remission of aggressive non-Hodgkins lymphoma (NHL). Repeated cycles of rIL-2, rIFN-alpha and histamine were administered to a patient with a grade III follicle center cell lymphoma, following relapse and high-dose chemotherapy with stem cell support. T-cell cytokine production and repertoire alterations were monitored by flow cytometry together with assessment of natural killer (NK) cell-mediated cytotoxicity. The treatment schedule induced significant increases in frequencies of CD4+ T cells expressing intracellular IFN-gamma or IL-4, thus a T helper (Th) 1 and Th 2 type of response were observed. CD8+T cells showed enhancement mainly of TNF-alpha production. Such induction of T-cell effector functions was accompanied by an augmentation of NK-cell cytotoxicity and a pronounced reduction of possibly regulatory CD57 expressing lymphocytes. The results indicate synergistic T- and NK-cell activation by tolerable doses of the combined immunotherapy, administered during remission after chemotherapy and ASCT in NHL.

Impaired cathepsin L gene expression in skeletal muscle is associated with type 2 diabetes.

To identify abnormally expressed genes associated with muscle insulin resistance or type 2 diabetes, we screened the mRNA populations using cDNA differential display combined with relative RT-PCR analysis from muscle biopsies of diabetes-prone C57BL/6J and diabetes-resistant NMRI mice fed with a high-fat or normal diet for 3 or 15 months. Six abnormally expressed genes were isolated from the mice after a 3-month fat feeding; one of them was cathepsin L. No significant difference in mRNA levels of these genes was observed between fat- and normal-diet conditions in either strains. However, cathepsin L mRNA levels in muscle were higher in normal diet-fed C57BL/6J mice compared with normal diet-fed NMRI mice at 3 months (0.72 +/- 0.04 vs. 0.51 +/- 0.04 relative units, P < 0.01, n = 8-10) and at 15 months (0.41 +/- 0.05 vs. 0.27 +/- 0.04 relative units, P = 0.01, n = 9-10). Further, cathepsin L mRNA levels in muscle correlated inversely with plasma glucose in both strains regardless of diets at 3 (r = -0.49, P < 0.01, n = 31) and 15 (r = -0.42, P = 0.007, n = 39) months. To study whether cathepsin L plays a role in human diabetes, we measured cathepsin L mRNA levels in muscle biopsies taken before and after an insulin clamp from 12 monozygotic twin pairs discordant for type 2 diabetes and from 12 control subjects. Basal cathepsin L mRNA levels were not significantly different between the study groups. Insulin infusion increased cathepsin L mRNA levels in control subjects from 1.03 +/- 0.30 to 1.90 +/- 0.32 relative units (P = 0.03). Postclamp cathepsin L mRNA levels were lower in diabetic twins but similar in nondiabetic twins compared with control subjects (0.66 +/- 0.22, 1.16 +/- 0.18 vs. 1.38 +/- 0.21 relative units, P < 0.02, NS, respectively). Further, postclamp cathepsin L mRNA levels were correlated with insulin-mediated glucose uptake (r = 0.37, P = 0.03), particularly, with glucose oxidation (r = 0.37, P = 0.03), and fasting glucose concentrations (r = -0.45, P < 0.01) across all three study groups. In conclusion, muscle cathepsin L gene expression is increased in diabetes-prone mice and related to glucose tolerance. In humans, insulin-stimulated cathepsin L expression in skeletal muscle is impaired in diabetic but not in nondiabetic monozygotic twins, suggesting that the changes may be secondary to impaired glucose metabolism.

Fat feeding impairs glycogen synthase activity in mice without effects on its gene expression.

To examine whether the effects of high-fat feeding on glycogen synthase (GS) activity and mRNA levels differ between diabetes-prone (C57BL/6J) and diabetes-resistant mice (NMRI), we measured GS activity and mRNA levels in muscle from C57BL/6J and NMRI mice fed a high-fat or normal chow diet for 3, 6, and 15 months. As compared with chow feeding, fat feeding increased plasma insulin levels in C57BL/6J mice at 15 months (464 +/- 29 v 267 +/- 47 pmol/L, P =.005), which was associated with elevated plasma glucose levels at 15 months (5.3 +/- 0.3 v 3.8 +/- 0.2 mmol/L, P =.001). Fat feeding increased plasma insulin levels also in NMRI mice at 15 months (705 +/- 145 v 275 +/- 64 pmol/L, P =.01) without, however, a rise of plasma glucose levels. In parallel with increased insulin levels, decreased muscle GS fractional velocity (FV) was observed at 6 (49.0% +/- 2.6% v 69.1% +/- 7.3%, P =.04) and 15 (45.8% +/- 1.8% v 53.4% +/- 1.6 %, P <.01) months but not at 3 months in the fat-fed C57BL/6J mice. Similarly, there was a significant decrease in GS fractional activity at 3 (57.9% +/- 4.3% v 70.4% +/- 2.6 %, P <.03) and 15 (47.3% +/- 2.4% v 56.4% +/- 2.1%, P =.02) but not at 6 months in the fat-fed NMRI mice. The decrease in GS activity was not associated with changes in mRNA levels at any time points. We conclude that (1) fat feeding results in similar elevation of plasma insulin levels and impairs GS activity in C57BL/6J and NMRI mice, and (2) the changes in GS activity do not involve effects on gene expression.

Down-regulation of insulin receptor substrates (IRS)-1 and IRS-2 and Src homologous and collagen-like protein Shc gene expression by insulin in skeletal muscle is not associated with insulin resistance or type 2 diabetes.

To examine whether altered gene expression of insulin receptor substrates (IRS)-1 and IRS-2 and Src homologous and collagen-like protein Shc is an inherited trait and is associated with muscle insulin resistance or type 2 diabetes, we measured mRNA levels of these genes by a relative quantitative RT-PCR method in muscle biopsies taken before and after an insulin clamp from 12 monozygotic twin pairs discordant for type 2 diabetes and 12 control subjects. Insulin-stimulated glucose uptake was decreased both in the diabetic and nondiabetic twin, compared with healthy control subjects (5.2 +/- 0.7 and 8.5 +/- 0.8 vs. 11.4 +/- 0.9 mg/kg x min(-1); P < 0.01 and P < 0.02, respectively). Basal mRNA levels of IRS-1, IRS-2, and Shc were similar in the diabetic and nondiabetic twins as well as in the control subjects. Insulin decreased mRNA expression of IRS-1 by 72% (from 0.75 +/- 0.06 to 0.21 +/- 0.04 relative units; P < 0.001), IRS-2 by 71% (from 0.55 +/- 0.10 to 0.16 +/- 0.08 relative units; P < 0.03), and Shc by 25% (from 0.95 +/- 0.04 to 0.71 +/- 0.04 relative units; P < 0.01) vs. baseline as demonstrated in the control subjects. The postclamp Shc mRNA level was slightly higher in the diabetic twins (P = 0.05) but similar in the nondiabetic twins, as compared with the control subjects, whereas postclamp IRS-1 and IRS-2 mRNA levels were similar between the study groups. There was an inverse correlation between postclamp Shc mRNA concentration and glucose uptake (r = -0.53, P = 0.01; n = 22) in the controls and nondiabetic twins. However, the decrease in Shc gene expression by insulin was not significantly different between the study groups. In conclusion, because insulin down-regulates IRS-1, IRS-2, and Shc gene expression in skeletal muscle in diabetic and nondiabetic monozygotic twins and control subjects to the same extent, it is unlikely that expression of these genes is an inherited trait or contributes to skeletal muscle insulin resistance.