Use Of Human Plasma Research Articles

Biological properties and characterization of several variations of a clinical human plasma-based skin substitute model and its manufacturing process.

Human plasma is a natural biomaterial that due to their protein composition is widely used for the development of clinical products, especially in the field of dermatology. In this context, this biomaterial has been used as a scaffold alone or combined with others for the development of cellular human plasma-based skin substitutes (HPSSs). Herein, the biological properties (cell viability, cell metabolic activity, protein secretion profile and histology) of several variations of a clinical HPSS model, regarding the biomaterial composition (alone or combined with six secondary biomaterials - serine, fibronectin, collagen, two types of laminins and hyaluronic acid), the cellular structure (trilayer, bilayer, monolayer and control without cells) and their skin tissue of origin (abdominal or foreskin cells) and the manufacturing process [effect of partial dehydration process in cell viability and comparison between submerged (SUB) and air/liquid interface (ALI) methodologies] have been evaluated and compared. Results reveal that the use of human plasma as a main biomaterial determines the in vitro properties, rather than the secondary biomaterials added. Moreover, the characteristics are similar regardless of the skin cells used (from abdomen or foreskin). However, the manufacture of more complex cellular substitutes (trilayer and bilayer) has been demonstrated to be better in terms of cell viability, metabolic activity and wound healing protein secretion (bFGF, EGF, VEGF-A, CCL5) than monolayer HPSSs, especially when ALI culture methodology is applied. Moreover, the application of the dehydration, although required to achieve an appropriate clinical structure, reduce cell viability in all cases. These data indicate that this HPSS model is robust and reliable and that the several subtypes here analysed could be promising clinical approaches depending on the target dermatological disease.

A new methodology combining QCM-D and proteomic profiling enables characterization of protein adsorption on 2D surfaces.

One of the critical features of biomedical material design is controlling the plasma protein adsorption to modulate the material behavior in biological media. Protein adsorption is highly influenced by the material surfaces and the proteins present in the biological medium. Thus, it is necessary to study protein-surface interactions that eventually take place on nanomaterials introduced into the body by the use of human plasma. However, very little information is available about human plasma interaction with planar surfaces under physiological conditions. Due to the limitation of the current characterization techniques to investigate the complicated interaction between the complex milieu of plasma proteins and planar materials, most efforts have focused on single proteins. To face this challenge, we have developed a new methodology based on the combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and liquid chromatography coupled with mass spectrometry (LC-MS) to obtain information about protein-surface interactions on planar surfaces. First, QCM-D allowed us to determine the adsorbed protein mass and layer thickness. After detaching the proteins by a surfactant treatment, LC-MS analysis revealed the proteomic profile. Here, we have investigated three base materials, polystyrene (PS), gold (Au), and silica (SiO2) with or without precoating and compared the protein profiles.

Improved ELISA for linoleate-derived diols in human plasma utilizing a polyHRP-based secondary tracer.

Dihydroxyoctadecenoic acids (DiHOMEs) are cytochrome P450 pathway-derived metabolites of linoleic acid, a highly abundant dietary fatty acid. They serve thermogenic functions at low concentrations but, at high concentrations, are involved in proinflammatory and deleterious outcomes in a wide range of pathologies. Hence, the development of a reliable analytical method is critical to elucidate their potential as biomarkers of health, and enzyme-linked immunoassay (ELISA)-based approaches offer unique benefits as alternatives to traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Accordingly, an earlier ELISA for DiHOMEs was dramatically improved employing new secondary tracers and geared towards use in human plasma, a universal matrix in biomedical applications, as well as urine. Three ELISA formats, two utilizing polyHRP-based secondary labels for signal amplification, were compared. The best format involved a biotinylated detection antibody and a polyHRP-conjugated streptavidin tracer. Assay detectability was enhanced 20-fold, relative to the original immunoassay, and performance assessments validated precision, selectivity, and robustness. Fast and easy extraction-clean up steps yielded high analytical recovery and permitted the assay to operate in moderate concentrations (up to 20%) of plasma, expanding its practical relevance. Finally, the ELISA was applied towards detection of DiHOMEs in clinical samples and authenticated with complementary LC-MS/MS analysis. Hence, the method provides a valuable analytical tool to investigate the diverse and extensive roles of DiHOMEs in regulatory biology.

Development and validation of an LC-MS/MS method for the quantitative analysis of milciclib in human and mouse plasma, mouse tissue homogenates and tissue culture medium

Milciclib is a promising cyclin-dependent kinase inhibitor currently in phase II clinical trials to treat several types of cancer. The first bioanalytical method for the quantitative analysis of milciclib in several biomatrices using liquid chromatography-tandem mass spectrometry is described here. This method was fully validated in human plasma according to FDA and EMA guidelines, and partially validated in mouse plasma, homogenates of mouse brain, kidney, liver, small intestine, spleen, and tissue culture medium. Palbociclib, an analog compound, was used as internal standard. A simple and fast sample pre-treatment by protein precipitation with acetonitrile was used, leading to efficient extraction of the analyte with recoveries between 95–100%. Chromatographic separation was achieved with a C18 analytical column and a gradient elution using 10 mM ammonium bicarbonate in water and 10 mM ammonium bicarbonate in water-methanol (1:9, v/v). This assay was selective, accurate, precise and linear in the concentration range of 1−1000 ng/mL. Moreover, samples above the upper limit of quantification can be integrally diluted up to 10-fold prior to analysis. The use of human plasma as a surrogate matrix to quantify milciclib in tissue culture medium and mouse matrices resulted in acceptable accuracy and precision, however tissue culture medium samples required a dilution with human plasma prior the pre-treatment. All performance parameters of the method complied with the acceptance criteria recommended by the guidelines, except for the carry-over, which was slightly above (22.9% of the lower limit of quantification) the recommended percentage (20%). Therefore, additional measures were taken to assure data integrity. Stability of milciclib in all matrices was evaluated, and in some matrices the analyte was unstable under the tested conditions. It is therefore recommended to keep these samples as briefly as possible at room temperature during the pre-treatment, and to store them at –70 °C to avoid analyte degradation. This method was successfully applied to support preclinical pharmacokinetic studies of milciclib.

New process for purifying high purity α1-antitrypsin from Cohn Fraction IV by chromatography: A promising method for the better utilization of plasma.

α1-antitrypsin (AAT) is a 52kDa serine protease inhibitor that is abundant in plasma. It is synthesized mainly by hepatic cells, and widely used to treat patients with emphysema due to congenital deficiency of AAT. A new isolation method for the purification of AAT from Cohn Fraction IV (Cohn F IV) is described. Cohn F IV is usually discarded as a byproduct from Cohn process. Using Cohn F IV as starting material does not interfere with the production of other plasma proteins and the cost of purification could be reduced greatly. Parameters of each step during purification were optimized, 15% polyethyleneglycol (PEG) concentration and pH 5.2 for PEG precipitation, elution with 0.05M sodium acetate and pH 4.7 for ion-exchange chromatography, and two steps blue sepharose affinity chromatography were chosen for AAT purification. The final protein with purity of 98.17%, specific activity of 3893.29 IU/mg, and yield of 28.35%, was achieved. Western blotting was applied for qualitative identification of final product, which specifically reacted with goat anti-human AAT antibody. LC-ESI-MS/MS was also employed to confirm the final protein. High performance liquid chromatography was used to analyze the composition of purified protein suggesting that pure protein was achieved. The molecular weight of AAT is 51062.77Da which was identified by LC-MS-MS. The manufacturing process described here may make better use of human plasma with Cohn F IV as starting material. The simple process described in this study is simple and inexpensive, it has a potential value for large scale production.

Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII.

Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generation of pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production with the use of BDDrFVIII as an immunogen. The main immunochemical characteristics of the produced antibodies and their potential biomedical applications are also reported. Rabbits were immunized with BDDrFVIII as an emulsion with Freund's adjuvant or with antigen immobilized in polyacrylamide gel (PAAG). Antibody titers in immune sera were assayed by enzyme-linked immunosorbent assay (ELISA). IgG purification was performed by afine chromatography on protein A-sepharose. Immune sera and IgG were tested by immunoblotting with the use of human plasma of healthy donors and people with hemophilia A, platelet lysates, and commercial plasma-derived concentrates as sources of FVIII-related antigens. FVIII-producing human umbilical vein cells were processed for immunocytochemical staining with the use of purified anti-FVIII-antibodies. Immunization of rabbits with PAAG-trapped antigen induced more potent immune response compared to the standard immunization procedure with Freund's adjuvant. The lowest working amount of immune IgG, measured by ELISA, was ~50 ng. Immunoblotting demonstrated that anti-BDDrFVIII antibodies effectively recognize the whole FVIII molecule (320 kDa), as well as different truncated polypeptides thereof, and are suitable for immunocytochemical analysis of FVIII-producing cells. An optimized procedure for the production of polyclonal antibodies against FVIII with the use of PAAG-immobilized BDDrFVIII (moroctocog alpha) was proposed and successfully validated. The produced antibodies are suitable for detecting and measuring FVIII-related antigens and may have various biomedical applications.

Blood Pressure, Heart Rate, and Urinary Catecholamines in Healthy Dogs Subjected to Different Clinical Settings

Correct interpretation of blood pressure (BP) and heart rate (HR) recordings is important in a clinical environment, but little is known about effects of stress on BP and HR responses of dogs to different clinical settings. To investigate BP and HR responses in different clinical settings in dogs of 3 breeds, and to relate findings to urinary catecholamine concentrations measured by ELISA assays previously validated for use in human plasma and urine, after validation for use in dogs. Client-owned healthy dogs; 41 Labrador Retrievers, 33 Cavalier King Charles Spaniels (CKCS), and 15 Dachshunds. Prospective observational study. BP and HR were measured in 4 clinical settings with or without veterinarian and owner present. Urine samples were taken before and after examination. ELISA assays were validated for canine urine, and epinephrine/creatinine and norepinephrine/creatinine ratios were analyzed. BP and HR were higher when measured by veterinarian alone than when owner was present (P < .020). Urinary catecholamine/creatinine ratios were higher after examination, compared with before, in all dogs (P < .0001). Labrador Retrievers had lower diastolic BP than Dachshunds in 2 settings (P ≤ .041), lower HR than CKCSs in 3 settings (all P < .0001), and lower catecholamine/creatinine ratios after examination than both other breeds (P ≤ .035). The in-house validation showed mean spiked recovery of 96.5% for epinephrine and 83.8% for norepinephrine. BP and HR responses were related to breed as well as clinical setting. Breed differences were detected in urinary catecholamine/creatinine ratios. Further studies on breed differences are warranted.

Abstract 5163: Elevations of intracellular kinases AKT1, GSK3β, JNK1, JNK2 and ERK2 in primary breast cancer tissue quantified with a novel multi-panel of single molecule immunoassays

Abstract The MAPK and PI3K signaling cascades are critical pathways in malignant transformation, tumor progression and multi-drug resistance (MDR) of human cancers. Clinical investigations into the role of phosphorylation of specific kinases are hindered by the lack of ultra-sensitive assay technology with requisite isoform- and phophorylation-state specificity. Improved assays are necessary to determine clinical relevance and potential diagnostic utility with a high degree of specificity. We tested adjacent normal and primary breast cancer tissue lysates with a novel panel of ten immunoassays for quantifying the total and phosphorylated forms of AKT1, GSK3β, JNK1, JNK2 and ERK2. The ultra-sensitive assay panel was developed for use with the Erenna® Immunoassay System (Singulex), which uses single molecule detection technology. Analytical sensitivity, inter-assay precision, linearity and specificity were determined and compared to corresponding ELISA assays. The novel assay panel was validated in HeLa and Jurkat cell lysates, region of linearity was determined, and phospho-specificity of each assay was confirmed. All assays were validated for use in human plasma, cell culture lysates, and human tissue specimens. Results in matched breast cancer tissue lysates showed that all total kinase concentrations (tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2) were found to be elevated in primary tumors compared to surrounding adjacent normal tissue, however concentrations of phosphorylated forms (pAKT1, pGSK3β, pERK2, pJNK1 and pJNK2) were not. All ten novel assays displayed exceptional linearity (R2=0.99) and precision (%CV 4.1 – 9.6), and were significantly more sensitive than the corresponding ELISA assay method based upon sample endpoint dilutions. The detection limits of the tAKT1, pAKT1, tGSK3β, pGSK3β, pERK2, tJNK2 and pJNK2 assays were all ≤0.3 pg/mL, while the detection limit of tERK2 was &amp;lt; 2pg/mL, tJNK1 was 4.5 pg/mL, and pJNK1 was 7 pg/mL. The LLoQ of each assay in the panel was sufficient for quantification from a minimum of approximately 100-200 cells, with exception of the total JNK1 assay, which required a minimum of approximately 800 cells in order to be quantifiable. We show that significant elevations in total kinase concentrations of tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2 can be quantified in primary tumors compared to surrounding adjacent tissue using a panel of isoform- and phosphorylation-specific single molecule immunoassays. We have further demonstrated that assays in this panel can quantify analytes from as little as 100 cells, and exhibit superior analytical performance over ELISA based methods. Thus, these novel assays greatly improve upon pre-existing ELISA technology that is limited by assay sensitivity, providing a new tool for clinical investigations of the MAPK and PI3K signaling cascades. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5163. doi:10.1158/1538-7445.AM2011-5163

The use of human plasma as matrix for calibration standards in pre-clinical LC–MS/MS methods—A way to reduce animal use

The option, for practical and ethical reasons, to replace animal plasma with human plasma for calibration standards was successfully applied to 73 analytical methods developed in our laboratory during the last years. The animals used for obtaining blank plasma could then be reduced with a number corresponding to about 25% of mice or 5% of rats in ordinary one-month toxicology studies. This is of important public concern and also in accordance with the 3R-strategy. The methods were successfully validated for determination of drug concentrations in plasma from rat, dog, mouse, rabbit and cynomolgus monkey. Reproducibility of study samples from dosed animals was established, showing a mean accuracy of 100.8% with a CV of 7.2% (n=1339). The purpose of this paper is to present a scientific basis for the alternative approach to adopt human plasma matrix for calibration standards, which will reduce animal use, without compromising the quality of appropriately validated assays. Additional advantages are cheaper and simplified plasma maintenance and the possibility to validate methods for several species in the same analytical batch.

Increasing hybridoma viability and antibody repertoire after the cell fusion by the use of human plasma as an alternative supplement

Prion diseases such as Bovine Spongiform Encephalopathy (BSE) and new variant Creutzfeldt–Jakob disease (nvCJD) have caused a major safety concern in cell cultures using fetal calf serum (FCS). In this study, we found that screened and tested human plasma (HP) obtained from blood centers may be an ideal alternate nutrient substitute to FCS for culturing hybridoma. In addition to the inherent safety, a ten-fold increase in the fusion efficiency has been observed if the HP was used as the nutrient supplement instead of FCS. Subsequently, a broader antibody repertoire may be recovered. The HP supplement was found to promote the growth of hybridoma cells but no impact on antibody secretion. Interestingly, this effect of enrichment was only observed for HP, but not plasma from other animals. Unidentified murine hybridoma cloning factors other than IL-6 may specifically reside in human blood.

Sensitive and specific detection of α‐synuclein in human plasma

alpha-Synuclein (alphasyn) mutations, overexpression, misfolding, and aggregation are associated with Parkinson's disease. This protein has been intensively studied in neuronal systems. However, alphasyn is also present in extracellular fluids, such as cerebrospinal fluid and blood plasma. Recent studies have attempted to quantify its levels and compare these in various extracellular fluids of control and Parkinson's disease subjects. Data from these studies have been difficult to interpret, suggesting that more sensitive, standardized, and well-characterized assays of larger cohorts are required. Here, we describe the development of a new ELISA specifically for quantifying alphasyn in human plasma. An initial assay, using a commercial anti-alphasyn monoclonal antibody (211; Santa Cruz Biotechnology, Santa Cruz, CA) and based on a published protocol, was adapted for use in human plasma. In addition, we have developed a novel alphasyn-specific antibody for the assay that has very high sensitivity and signal:noise characteristics. Assays with either antibody showed high specificity for alphasyn, and detected it in a variety of sample types, including plasma. These assays can now be employed on large cohorts of patients and control subjects to determine whether plasma levels are altered in disease. Although measuring extracellular alphasyn levels may prove to be a useful biomarker of Parkinson's disease, it should also be a powerful tool for basic research aimed at understanding the normal and pathological physiology of alphasyn secretion. .

Abstract A26: Elevations of intracellular kinases AKT1, GSK3β, JNK1, JNK2, and ERK2 in primary breast cancer tissue quantified with a novel multipanel of single-molecule immunoassays

Abstract Purpose: The MAPK and PI3K signaling cascades are critical pathways in malignant transformation, tumor progression and multidrug resistance (MDR) of human cancers. Elucidation of the relative contributions of distinct kinases is made difficult due to many factors, including prevalence of multiple isoforms, posttranslational modifications, synergistic activation and pathway crosstalk. Clinical investigations into the role of phosphorylation of specific kinases are further hindered by the lack of ultrasensitive assay technology with requisite isoform- and phophorylation-state specificity. Improved assays are necessary to determine clinical relevance and potential diagnostic utility with a high degree of specificity. Methods: Adjacent normal and primary breast cancer tissue lysates were analyzed using a novel panel of ten immunoassays for quantifying the total and phosphorylated forms of AKT1, GSK3β, JNK1, JNK2 and ERK2. The ultrasensitive assay panel was developed for use with the Erenna® Immunoassay System (Singulex), which uses single molecule detection technology. Analytical sensitivity, interassay precision, linearity, and specificity were determined and compared to corresponding ELISA assays. The novel assay panel was validated in HeLa and Jurkat cell lysates, region of linearity was determined, and phospho-specificity of each assay was confirmed. All assays were validated for use in human plasma, cell culture lysates, and human tissue specimens. Results: Testing in matched breast cancer tissue lysates showed that all total kinase concentrations (tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2) were found to be elevated in primary tumors compared to surrounding adjacent normal tissue, however concentrations of phosphorylated forms (pAKT1, pGSK3β, pERK2, pJNK1 and pJNK2) were not. All ten novel assays displayed exceptional linearity (R2=0.99) and precision (%CV 4.1 – 9.6), and were significantly more sensitive than the corresponding ELISA assay method based upon sample endpoint dilutions. The detection limits of the tAKT1, pAKT1, tGSK3β, pGSK3β, pERK2, tJNK2 and pJNK2 assays were all ≤0.3 pg/mL, while the detection limit of tERK2 was &amp;lt; 2pg/mL, tJNK1 was 4.5 pg/mL, and pJNK1 was 7 pg/mL. The LLoQ of each assay in the panel was sufficient for quantification from a minimum of approximately 100 to 200 cells, with exception of the total JNK1 assay, which required a minimum of approximately 800 cells in order to be quantifiable. Conclusions: We show that significant elevations in total kinase concentrations of tAKT1, tGSK3β, tERK2, tJNK1 and tJNK2 can be quantified in primary tumors compared to surrounding adjacent tissue using a panel of isoform- and phosphorylation-specific single molecule immunoassays. We have further demonstrated that assays in this panel can quantify analytes from as little as 100 cells, and exhibit superior analytical performance over ELISA based methods. Thus, these novel assays greatly improve upon pre-existing ELISA technology that is limited by assay sensitivity, providing a new tool for clinical investigations of the MAPK and PI3K signaling cascades. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A26.

A pseudo‐outbreak of yeast in cell blocks associated with the use of human plasma

A pseudo‐outbreak of yeast in cell blocks associated with the use of human plasma

An Enantiomer-Selective Liquid Chromatography-Tandem Mass Spectrometry Method for Methadone and EDDP Validated for Use in Human Plasma, Urine, and Liver Microsomes

A liquid chromatography-electrospray ionization-tandem mass spectrometry method has been developed and validated to detect (R)- and (S)-methadone and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma with cross-validation to urine and liver microsomes. Use of deuterated internal standards and liquid-liquid extraction coupled with chiral separation provided baseline separation with a lower limit of quantitation (LLOQ) of 2.5 ng/mL. The LLOQ was established from comparison of signal in blanks from six different sources per matrix with the same sources fortified at the LLOQ (none exceeded 19% of LLOQ) and precision and accuracy at the LLOQ determined in the same six sources per matrix. The assay was precise (% coefficients of variation within 13.8%) and accurate (% targets within 15%) in all three matrices. No interference was seen from addition of other psychoactive drugs. Stability was determined in plasma (24 h at room temperature, 321 days at -20 degrees C, 3 freeze-thaw cycles); processed plasma samples (5 days at -20 degrees C, 12 days on autosampler); urine (24 h at room temperature); and stock solutions (20 h at room temperature, 61 days at -20 degrees C). Applications of varying degree are presented for each matrix. Plasma from five subjects maintained on 100 mg oral methadone per day permitted comparison of the pharmacokinetics of the enantiomers. The t(1/2) of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C(max), AUC, C(min), and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t(1/2) of (R)-EDDP was significantly greater than (S)-EDDP. In spot urines, (R)- was higher than (S)-methadone, and (S)- was generally higher than (R)-EDDP. (R)- and (S)-EDDP production was detected after incubation of therapeutic concentrations of racemic methadone with human liver microsomes, and (S)-EDDP production was twofold greater than (R)-EDDP in three human placental microsomes incubated with racemic methadone.

In vitro control of human bone marrow stromal cells for bone tissue engineering.

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.

Alternatives to animal sera for human bone marrow cell expansion: human serum and serum-free media.

The increasing use of cultured human cells in clinical trials is highlighting the need for alternatives to media containing animal sera that are typically used to support these cultures. Perfused cultures of BM mononuclear cells (MNC) were used to evaluate animal sera alternatives with respect to the output of primitive, progenitor, and stromal cells. A serum level of 20% was optimal, and this could be provided by FBS alone or by a mixture of horse serum (HoS) and FBS, but not by HoS alone. Allogeneic human plasma (20%) supported half the level of cell, CFU-GM, and LTC-IC output as compared with animal sera-containing control. Significant donor-to-donor variability in human plasma was observed, but this was mitigated by pooling of plasma samples. Autologous and allogeneic human plasma performed equivalently. The use of autologous or allogeneic human serum was found to be equivalent to the use of human plasma, but all were inferior to animal sera. Animal sera supported typical stroma and cobblestone formation, whereas stroma in human serum cultures was less dense. Eight commercial serum-free media were tested and found to support MNC expansion to varying degrees, but none approached the performance of the animal serum-containing control, particularly with respect to stromal (i.e., CFU-F) support. In fact, when MNC were cultured in parallel with CD34-enriched cells, output (from MNC) was higher only in control medium, apparently because serum-free media reduced accessory cell effects. Because of these results, a new serum-free medium was developed for MNC cultures. This formulation outperformed all commercial serum-free media, resulting in cell and LTC-IC output equivalent to that of control. However, CFU-GM and CFU-F output were 66% and 9% of control, respectively. Precoating the culture surface with collagen increased CFU-F (and Thy-1+ cell) output to control levels, although CFU-GM output was still lower than control. The addition of either fibronectin or PDGF had no measurable effect, nor did the use of 5-100-fold greater concentrations of growth factor supplementation. The serum-free medium also increased CD41+ and CD61+ cell output to 150%-220% of control levels. The development of this new serum-free medium has potential for use in the perfused BM MNC culture systems currently in clinical trials to test the efficacy of expanded cells after cytoablative chemotherapy.

12. Development of a crosslinked soluble fibrin assay for use in human plasma

12. Development of a crosslinked soluble fibrin assay for use in human plasma

Autologous Whole Plasma Fibrin Gel

Fibrin glue is a relatively recent addition to the armamentarium of hemostatic agents for surgical use. Its efficacy has been repeatedly demonstrated in almost all surgical disciplines and subspecialties. Its use in the United States has been limited because of the risk of viral transmission associated with the use of human plasma. Previous authors have described techniques that limit this risk, but they are frequently impractical, expensive, or cumbersome. We describe the use of patients' own fresh plasma to make fibrin gel at the operative field. It provided hemostasis at least as good as that from heterologous plasma glue in 40 cardiac surgical patients. Autologous whole plasma fibrin gel is inexpensive and safe and eliminates the risk of viral transmission associated with glue derived from heterologous donor plasma.

A Screening Test for Modified Hemoglobin Blood Substitutes Before Clinical Use: Based on C3a Complement Activation in Human Plasma

Modified hemoglobin preparations may potentially cause hypersensitivity and anaphylactic reactions, antibody-antigen reactions and other problems. Unfortunately response in animal safety studies may not reflect the same response in human. The next best test before clinical trial in human may the use of human plasma in-vitro. This paper present an in-vitro procedure based on complement activation (C3a) of human plasma. The procedure involves collecting heparinised blood and separating the plasma and freezing the heparinised plasma at -70 degrees C until use. Each 100 lambda of control or test samples is added to 400 lambda of this plasma. This is incubated at 37 degrees C, 60 rpm for 1 hour, then added to EDTA saline to stop the reaction and stored at -70 degrees C until analysed by standard radioimmunoassay for C3a. (C3 measurement is not sensitivity enough). Using the screening test procedure described above, C3a levels (ng/ml) in plasma were: control, 1,980 +/- 280; Zymosan, 20,000; Hemoglobin preparation A, 2,227 +/- 617; Hemoglobin preparation B, 4,967 +/- 153; A 75% + B 25%, 3,967 +/- 270; A 50% + B 50%, 4,553 +/- 517; A 25% + B 75%, 4,920 +/- 430. Hemoglobin preparation A did not cause significant increases in C3a complement activation. Hemoglobin preparation B caused significant increase in C3a complement activation. Serial dilution of Hemoglobin preparation B in Hemoglobin preparation A continued to cause the same degree of C3a complement activation. This is not due to C3 exhaustion because Zymosan resulted in C3a of greater than 20,000ng/ml. This appears to show that this screening test can detect complement activation even at low concentrations of the hemoglobin preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of human plasma for continuous in vitro cultivation of Plasmodium falciparum

Plasma from units of human blood collected in CPDA-1 preservative were compared with human serum in cultures of Plasmodium falciparum. No significant differences in parasite growth and multiplication were seen between cultures containing serum or plasma. The wider availability of plasma makes it an attractive alternative to serum, especially in large scale cultures.