Safety of platelet transfusion: past, present and future.

Platelet components became routinely available to many institutions in the late 1960s and since then utilization has steadily increased. Platelets are produced by three principal methods and their manufacturing process is regulated by multiple agencies. As the field of platelet transfusion has evolved, a broad array of strategies to improve platelet safety has developed. This review will explore the evolution of modern platelet component therapy, highlight the various risks associated with platelet transfusion and describe risk reduction strategies that have been implemented to improve platelet transfusion safety. In closing, the reader will be briefly introduced to select investigational platelet and platelet-mimetic products that have the potential to enhance platelet transfusion safety in the near future.

Transportation of cellular therapy products: report of a survey by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) collaborative.

BACKGROUND AND OBJECTIVES: Most cell therapy products (CTP) are infused or processed shortly after collection but in some cases this may be delayed for up to 48 h. A number of variables such as temperature and cell concentration are of critical importance for the integrity of CTP during this time. MATERIALS AND METHODS: We conducted a survey of cellular therapy laboratories to ascertain current practices for CTP transportation. RESULTS: There were 194 respondents of whom 90% shipped or received CTP--84% allogeneic, 71% autologous and 62% therapeutic cells. Processing facilities shipped or received the following products--hematopoietic progenitor cells (HPC), Marrow 73%; HPC, Apheresis 90%; HPC, Cord Blood 54% and others 14%. Other CTP included donor lymphocytes, mesenchymal stem cells (MSC), natural killer cells, buffy coat neutrophils and virus-specific cytotoxic T lymphocytes (CTL). More than 70% of respondents believed that it was acceptable for CTP to be held for up to 2 h without checking the temperature or cell density and a similar proportion agreed that putting products in containers to control parameters such as temperature within this time period was unnecessary. The majority of centres shipped or received between 1 and 10 CTP annually and 66% received products taking more than 2 h to ship. Of these, 82% specified the conditions for temperature in transit whilst 57% monitored temperature in transit and 74% of these used a data logger. The temperature range most commonly specified was 18-24 degrees C. The majority of processing facilities did not request an adjustment to the cell density even for products taking more than 2 h to reach their facility. More than 90% of respondents tested HPC for CD34(+) cells, viability and sterility; 40-48% performed colony-forming unit-granulocyte macrophage (CFU-GM) analysis. Only viability was thought by > 50% of respondents to be impacted by temperature, cell density and other parameters. CONCLUSION: Understanding current practice will help in the design of future studies for CTP storage and transportation.

Development of a clinical model for ex vivo expansion of multiple populations of effector cells for adoptive cellular therapy.

BACKGROUND: We have previously demonstrated a laboratory model for expanding autologous mononuclear cells into populations of effector killer cells. The goal of the current experiments was to develop a good manufacturing practice (GMP) method for expanding clinical-grade activated effector cells that mediate tumor cell killing through various mechanisms that could be infused into patients following high-dose chemotherapy and autologous stem cell transplant. METHODS: Mobilized mononuclear cells (MNC) from myeloma patients were placed in culture with serum-free AIM V media, interleukin-2 (1000 IU/mL) and OKT-3 (500 ng/mL) at 37 degrees C and 5% CO2. After 7 days of expansion, the cells were analyzed for cell concentration, viability, phenotype and cytotoxicity directed against human myeloma cell lines. Expansion was compared using culture bags and flasks. Cryopreserved expanded cells were also analyzed. RESULTS: This clinical model of ex vivo expansion yielded polyclonal populations of cytotoxic lymphocytes, including CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD8+ CD56+ T cells and CD56+ natural killer cells. Compared with flasks, culture bags provided a 2-3-fold effector cell expansion with minimal risk of contamination. The optimal cell concentration at the time of expansion was 2.5-3.5 x 10(6) peripheral blood MNC/mL. Viability and cytotoxicity were maintained if the expanded cells were cryopreserved and then thawed for use. DISCUSSION: The results demonstrate a reproducible and reliable GMP procedure that is currently being employed in a clinical trial. These expanded cells, and their various pathways of tumor cell killing, may circumvent tumor escape mechanisms and improve outcomes.

Multicenter study on in vitro characterization of dendritic cells.

BACKGROUND: There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability. METHODS: CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol. RESULTS: Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion. DISCUSSION: In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

Terminology and labeling of cellular products-2: Implementation plan.

The publication of new standards for terminology and labeling marks an important step in ensuring consistency and traceability of cellular therapies at the global level. However, it is only with the widespread implementation of the standard that the benefits can be truly realized. This paper provides guidance on the practical aspects of adopting these new standards for organizations with differing current levels of computerization. It discusses project management, equipment, licensing, and validation topics.

Terminology and labeling of cellular products: 1. Standards.

The International Cellular Therapy Coding and Labeling Advisory Group was established to address the growing need for standardization of terminology and labeling for cellular therapy products as a result of increasing international transfer of these products. This paper presents new standards for terminology and labeling. These standards have been developed through a consultative process and are supported by key professional and accreditation bodies. By using these standards, together with the unique donation identification numbers and international product reference tables provided by the International Society of Blood Transfusion (ISBT) 128 Standard, consistency and traceability can be assured at the global level. A companion paper provides guidance on the implementation of the ISBT 128 system.

Early recovery of aggressive cytotoxic cells and improved immune resurgence with post-transplant immunotherapy for multiple myeloma.

A phase I/II trial evaluated early administration and dose escalation of interleukin (IL)-2 with granulocyte macrophage colony stimulating factor (GM-CSF) post-transplant. Following melphalan (200 mg/m(2)) and an autologous transplant, IL-2 was initiated (day 0) and continued for 4 weeks. GM-CSF (250 mcg/m(2)/day) began on day 5. Fifteen of 19 patients completed therapy. No treatment-related deaths occurred. IL-2 (1 x 10(6) IU/m(2)/day) was not tolerated in two of six patients due to > or =grade 3 fatigue/diarrhea (n=1) or supraventricular tachycardia (n=1). The maximum tolerated dose of IL-2 was 6 x 10(5) IU/m(2)/day; this dose was well tolerated by 11 of 13 patients. Neutrophil and platelet engraftment occurred on day 13 (median; range 10-17 days) and day 13 (median; range 0-74 days), respectively. When compared to control patients, there was a marked increase in the number of CD3+ T cells (P=0.005), CD4+ T cells (P=0.01), CD8+ T cells (P=0.001) and CD4+CD25+Treg cells (P=0.015) post-transplant. Cytotoxicity directed against myeloma cells was markedly increased when compared to control patients (P=0.017). This unique trial design using early administration of IL-2 with GM-CSF during the period of lymphodepletion, demonstrated a marked increase in the number and function of early cytotoxic effector T cells, without suppression of engraftment.

The generation of narrative interpretations in laboratory medicine: a description of service-specific sign-out rounds.

The logistical details for organizing effective interpretive rounds in a laboratory medicine subspecialty must be carefully established so that expert opinions are provided in a timely fashion in a patient-specific report, rather than as a collection of fixed comments associated with a particular laboratory result generated by a computer This report describes the test batteries for interpretations, the billing for interpretations, clinical examples of interpretations, and interpretations for which billing is not typically performed in several clinical or laboratory areas in our institution. These include coagulation disorders, hemoglobin and anemia evaluations, autoimmune disorders, serum protein analysis, toxicology, molecular diagnostics, and transfusion medicine. The information in this report should provide sufficient detail to allow development of interpretive services with successful billing for the areas in laboratory medicine described.

Collagen-induced whole blood platelet aggregation in patients undergoing surgical procedures associated with minimal to moderate blood loss.

Intraoperative bleeding due to platelet disorders is a persistent problem. Therefore, a screening assay to identify patients who are likely to bleed as a result of platelet dysfunction would be useful in formulating decisions about patient care. A previous study indicated that preoperative collagen-induced whole blood platelet aggregation predicts bleeding in patients undergoing surgery with cardiopulmonary bypass, a procedure associated with substantial blood loss. In the current study, we assessed the ability of the same whole blood platelet aggregation test to predict blood loss in patients undergoing surgical procedures not associated with substantial blood loss. The study included 369 adult patients (165 men and 204 women). Patients were categorized in three groups depending on the invasiveness of the operation and the expected blood loss. The intraoperative estimated blood loss value, obtained from the operative report in the patient record, increased significantly with increasing surgical invasiveness. Patients with excessive blood loss (defined as blood loss at or above the 75th or 90th percentile of the estimated blood loss values of patients undergoing procedures of similar invasiveness) had similar platelet aggregation values as patients who did not experience excessive blood loss. Thus, for patients undergoing operations not associated with substantial blood loss, the results of preoperative collagen-induced whole blood platelet aggregation are not effective in identifying patients likely to experience excessive blood loss.

Low-density lipoprotein reconstituted with fatty acid ethyl esters as a physiological vehicle for ethyl ester delivery to intact cells.

Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been found selectively in the organs damaged by ethanol abuse, and on that basis have been implicated as contributors to ethanol-induced organ damage. To directly assess the cytotoxic potential of FAEEs with intact cells in a physiological system, solubility must be achieved for these highly nonpolar lipids in aqueous medium. After ethanol ingestion, FAEEs can be found within low-density lipoproteins (LDLs). Therefore, to achieve solubility with FAEEs bound to a naturally occurring lipid carrier, we developed a method for FAEE solubilization and delivery to cells in culture. We synthesized radiolabeled FAEEs and incorporated them into human LDL particles that bind to LDL receptors and deliver FAEEs to intact cells. Ethyl palmitate and ethyl oleate were incorporated into LDLs yielding molar ratios of FAEEs to LDLs of 2,153 +/- 249 and 4,208 +/- 403, respectively. LDL reconstituted with FAEE had the same electrophoretic mobility on agarose gel electrophoresis as native LDL, indicating that the reconstituted LDL (rLDL) was not oxidatively modified. Quantitative analysis of the solubilization of FAEEs in aqueous medium was investigated by adding FAEEs to tissue culture medium either directly or reconstituted in LDL at a concentration of 27 microM. The percentage of FAEE quantitated was 40.0 +/- 2.5% and 89.3 +/- 0.6% for FAEEs added directly and in rLDLs, respectively. After sterile filtration of these two media, the percentage of FAEE that remained was 11.8 +/- 1.3% (direct addition) and 74.9 +/- 1.3% (addition within rLDL), further demonstrating that the LDL particle did solubilize the FAEE.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acid ethyl esters decrease human hepatoblastoma cell proliferation and protein synthesis.

BACKGROUND/AIMS: Fatty acid ethyl esters (FAEEs) are nonoxidative products of ethanol metabolism. They have been implicated as mediators of ethanol-induced organ damage because FAEE and FAEE synthase have been found specifically in the organs damaged by ethanol abuse. This study showed toxicity specifically related to FAEE or their metabolites for intact human hepatoblastoma-derived cells (HepG2). METHODS: The lipid core of human low-density lipoprotein (LDL) was extracted and the LDL particle reconstituted with either ethyl oleate or ethyl arachidonate. Cultured HepG2 cells were incubated with LDL containing FAEE. Cell proliferation was measured by [methyl-3H]thymidine incorporation. Protein synthesis was determined using L-[35S]methionine. RESULTS: Incubation of cells with 600 mumol/L ethyl oleate or 800 mumol/L ethyl arachidonate decreased [methyl-3H]thymidine incorporation into HepG2 cells by 31% and 37%, respectively. LDL reconstituted with 400 mumol/L ethyl oleate decreased protein synthesis in intact HepG2 cells by 41%. Electron microscopy revealed significant changes in cell morphology, particularly involving the cell nucleus. FAEE delivered in reconstituted LDL were rapidly hydrolyzed and the fatty acids re-esterified into phospholipids, triglycerides, and cholesterol esters, with preference for triglycerides. CONCLUSIONS: These findings provide evidence that FAEE are toxic for intact human hepatoblastoma cells and that they or their metabolites may be an important causative agent in ethanol-induced liver damage.

Mode of transport of fatty acid to endothelial cells influences intracellular fatty acid metabolism.

Fatty acids are transported to cells from a variety of different moieties in the plasma. In this study, using oleate and human umbilical vein endothelial cells, we asked whether the vehicle that delivers fatty acid to cells has an influence on its metabolism upon its incorporation into the cell. For oleate vehicles, we compared free oleate bound to albumin with oleate in low density lipoprotein (LDL) which was delipidated and reconstituted with either radiolabeled triolein or cholesteryl oleate. Using approximately physiologic concentrations of LDL and free oleate, we demonstrated by three lines of evidence unique patterns of cellular oleate metabolism for oleate delivered as triolein within LDL, for oleate delivered as cholesteryl oleate within LDL, and for oleate delivered as free oleate bound to albumin. In fact, the difference was most marked between cholesteryl oleate and triolein, even though the oleate in cholesteryl oleate and triolein was delivered in identically reconstituted LDL particles, which were presumably incorporated into the cells and degraded in lysosomes in a similar fashion. First, we demonstrated that oleate delivered as free oleate or as triolein in reconstituted LDL was desaturated and elongated to fatty acid metabolites, but cholesteryl oleate in reconstituted LDL was not similarly metabolized. The elongated and desaturated metabolites of oleate were preferentially esterified in cellular triglyceride when oleate was delivered as free oleate, but they were preferentially esterified in phospholipids when oleate was delivered as triolein in LDL. Second, we observed that there was a difference in the distribution of oleate among phospholipids when oleate was delivered as cholesteryl oleate in reconstituted LDL versus triolein in reconstituted LDL. When the oleate was delivered as triolein in reconstituted LDL, there was greater esterification in diacyl phosphatidylethanolamine, in phosphatidylserine, and in phosphatidylinositol. When oleate was delivered as cholesteryl oleate in reconstituted LDL, there was greater esterification in diacyl phosphatidylcholine. Third, there was a marked preference for oleate delivered from triolein in LDL over cholesteryl oleate in LDL for esterification into the sn-1 position of plasmalogens as a vinyl ether-linked fatty acid. These data indicate that mode of transport of fatty acid to cells influences fatty acid metabolism upon its incorporation into the cell, even when the fatty acid is delivered from the core of the same lipoprotein.

Fatty acid ethyl esters: non-oxidative metabolites of ethanol.

Fatty acid ethyl esters are esterification products of fatty acids and ethanol. These compounds have been detected in the serum and cells of individuals following ethanol ingestion. Fatty acid ethyl esters can be quantitated by gas chromatography-mass spectroscopy (GC-MS) in the serum following ethanol ingestion and have been found in concentrations up to 42 microM. Fatty acid ethyl esters have also been isolated from adipose tissue of subjects ingesting fatty acid ethyl ester capsules as well as from subjects ingesting ethanol. HepG2 cells, a human hepatoblastoma cell line, have also been shown to generate fatty acid ethyl esters when incubated with 1.25 microM fatty acid and 0.17 M ethanol. Fatty acid ethyl esters were found to be toxic to HepG2 cells when presented to the cells in the core of low density lipoprotein particles.

Induction of ornithine decarboxylase in normal and protein kinase C--depleted human colon carcinoma cells.

To examine the role of protein kinase C (PKC) in induction of human colon adenocarcinoma cell line, DETA/W, by polypeptide growth-promoting factors, ornithine decarboxylase activity (ODC) and DNA synthesis were determined in cells depleted of PKC. PKC depletion was achieved by prolonged cultivation (more than 30 passages) with 10(-6) M phorbol 12-myristate 13-acelate. Lack of PKC in studied cells was proved by measurements of PKC activity and immunoreactivity. Although ODC activities and DNA syntheses in PKC-depleted cells were decreased by about 40-50% compared to normal DETA/W cells, the percentage increase of these mitogen-responsive reactions was quantitatively similar in both cell sublines. These results raise the possibility that not all of the biological responses to growth factors are connected with the activation of calcium-dependent PKC.