Joint detection of important biomarkers and optimal dose-response model using penalties.

We propose a method to jointly detect influential biomarkers and estimate how they change with dose. The assessment is made in dose-ranging trials collecting multiple outcomes for efficacy, safety, pharmacokinetics or pharmacodynamics. We regress all these outcomes versus a non-parametric transformation of the dose. The regression coefficients and the parameters from the dose-response model are simultaneously estimated using a penalized alternating least-squares method. We illustrate the technique with a phase I clinical trial and a metabonomic experiment in rats.

A rapid and reliable flow cytometric routine method for counting leucocytes in leucocyte-depleted platelet concentrates.

BACKGROUND AND OBJECTIVES: To ensure a proper quality control it is important to use a reliable method to count low numbers of leucocytes in leucocyte-reduced platelet concentrates (PCs). MATERIALS AND METHODS: A modified flow cytometric method for counting low numbers of leucocytes, based on a reference population contained in tubes with an exact number of fluorescent beads and staining with propidium iodide was used. To increase the number of events, the original sample volume was increased. RESULTS: There was a good correlation in the number of leucocytes (r = 0.99) between the modified flow cytometric method and microscopy of samples from unfiltered and expected numbers from serially diluted PCs. Samples from leucocyte-reduced PCs obtained by apheresis or filtered buffy coats showed no correlation between results from the modified flow cytometric method and microscopy (Nageotte). CONCLUSION: Counting by microscopy gave a lower number of leucocytes than the modified flow cytometric method when counting a low number of cells. However, analysis of the serially diluted PCs proved that the modified flow cytometric method was reliable and rapid, making it suitable for clinical routine use.

White blood cell subsets in buffy coat-derived platelet concentrates: the effect of pre- and poststorage filtration.

BACKGROUND AND OBJECTIVES: Our objective was to study the effect of storage time on the filtration of platelet concentrates (PCs). We compared the total number of white blood cells (WBC), as well as the distribution of WBC subsets, in units filtered before and after storage. MATERIALS AND METHODS: Buffy coat-derived PCs were filtered either fresh or after 5 days of storage, and total WBC were enumerated by flow cytometry. WBC subsets were analyzed by flow cytometry with three-color fluorescence. RESULTS: The total number of white cells before filtration was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. Although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in both fresh and stored units; the percentage of T cells was decreased, whereas the percentage of B cells and monocytes was increased after filtration. CONCLUSION: Our results suggest that prestorage WBC filtration of platelet concentrates is superior in reducing the absolute numbers of WBC. However, both pre- and poststorage WBC filtration significantly affect the proportions of WBC in the final product, decreasing the number of T cells while apparently increasing the proportion of MHC class II-positive cell populations.

Factors influencing white cell removal from red cell concentrates by filtration.

BACKGROUND: The preparation of blood components by hard centrifugation results in red cell concentrates with a small amount of plasma. The influence of various plasma factors, temperature, and storage time on white cell reduction by filtration was studied. STUDY DESIGN AND METHODS: Red cell concentrates were suspended in 100 mL of saline-adenine-glucose-mannitol (SAGMAN) solution or in SAGMAN solution in which 5 or 10 mL had been replaced with an equal amount of fresh plasma, albumin (4%), or heat-inactivated plasma. After overnight storage at 4 degrees C, filtration at a slow flow rate (2 hours) was performed. The effect of temperature was studied by filtration at 4 degrees C and 37 degrees C. To study the influence of storage time, red cell concentrates were stored for 4 to 8 hours or 14 to 20 hours at 4 degrees C and filtered through another model of filter. The number of white cells was counted microscopically or by flow cytometry. RESULTS: When 5 or 10 mL of plasma was added, a significantly smaller number of white cells were found after filtration than were found in the SAGMAN control (the median difference between pairs: 23.6 x 10(6) for 5 mL [p = 0.006] and 14.9 x 10(6) for 10 mL [p = 0.003]). The number of white cells was significantly higher with 10 mL of albumin than with 10 mL of plasma (difference, 15.0 x 10(6); p = 0.006). When heat-inactivated plasma was used, the number of white cells was significantly lower than when fresh plasma was used (difference, 0.3 x 10(6); p = 0.009). Filtration at 37 degrees C resulted in a 64-percent reduction in white cells and that at 4 degrees C led to a 99.7-percent reduction (p = 0.006). When the second filter was used, a slight but significantly lower number of white cells was found in the red cell concentrate stored for 14 to 20 hours than in that stored for 4 to 8 hours (difference, 0.03 x 10(6); p < 0.001). CONCLUSION: The amount of plasma in the red cell concentrate and the storage time and temperature are important factors in the outcome of white cell reduction by filtration. The effect of plasma does not seem to be due to a general influence of protein or to the activity of complement or fibrinogen.

Growth factor release during preparation and storage of platelet concentrates.

The platelet content of platelet-derived growth factor (PDGF), a mitogen stored in the alpha-granules, was studied during preparation and storage of platelet concentrates (PC) and compared to the growth-promoting activity of platelets, beta-thromboglobulin (beta-TG) and lactate dehydrogenase (LD). We compared PC prepared from platelet-rich plasma (PRP-PC; n = 10) and from buffy coat. Two different pre-preparation storage periods of the buffy coat were used: 4 h (BC-PC:4h; n = 10) and 24 h (BC-PC:24h; n = 5). The platelet content of PDGF and beta-TG was measured by a RIA technique and the growth-promoting activity by incorporation of 3H-thymidine in stimulated fibroblasts. The platelet content of PDGF, beta-TG and the growth-promoting activity of the platelets decreased in a similar way during preparation and storage of PRP-PC (31 +/- 2, 35 +/- 2 and 33 +/- 7%, respectively, at day 5 of storage; mean +/- SEM). The release of LD was minor (3.9 +/- 0.5% at day 5). At day 1 of storage the platelet content of PDGF was significantly better preserved in BC-PC:4h than in BD-PC:24h (88 +/- 2 and 81 +/- 3%, respectively; p = 0.03). Comparing BC-PC:4h and PRP-PC we found a significantly better preservation of PDGF in BC-PC:4h until day 3 of storage (80 +/- 2 and 75 +/- 1%, respectively at day 3; p = 0.046). In conclusion the preparation of PC according to the PRP method initially induces a higher loss of PDGF, and hence of the growth-promoting activity, than the BC method.

Inadequate white cell reduction by bedside filtration of red cell concentrates.

BACKGROUND: White cell filtration of red cell concentrates is often performed at the bedside, in the ward, with the filter inserted in the blood administration line. The aim of this study was to evaluate the efficiency of this filtration method and compare it to filtration in the blood bank. STUDY DESIGN AND METHODS: One-day-old, buffy coat-reduced, hard-packed red cell concentrates in saline-adenine-glucose-mannitol solution were filtered through different filters designed for bedside or laboratory use. With filters designed for bedside use, filtration of red cells was performed under laboratory conditions at fast flow (10 min) or under bedside conditions at slow flow (2 hours). The remaining white cells were counted microscopically. Filters designed for laboratory use were evaluated at fast flow, and the number of contaminating white cells was counted by flow cytometry. RESULTS: With bedside filters, a significantly higher contamination of white cells was found in the units filtered at slow flow than at fast flow, regardless of the filter used. The number of units with > 5 x 10(6) white cells was 52 (78%) of 67 filtered at slow flow compared to 11 (23%) of 47 at fast flow, all filters taken together. This difference in white cell contamination was mainly due to an increase of polymorphonuclear cells in the red cell concentrates filtered at slow flow. With filters designed for laboratory use, 0 to 2 percent of units (n = 1448) were contaminated with > 5 x 10(6) white cells. CONCLUSION: Bedside filtration for white cell reduction at slow flow is inefficient for 1-day-old, buffy coat-reduced red cell concentrates.