Batches Of Serum Research Articles

Natural infection of cattle with an atypical `HoBi'-like pestivirus – Implications for BVD control and for the safety of biological products

During a study on Bovine Viral Diarrhoea (BVD) epidemiology in Thailand, a pestivirus was detected in serum from a calf. Comparative nucleotide sequence analysis showed that this virus was closely related to a recently described atypical pestivirus (D32/00_'HoBi') that was first isolated from a batch of foetal calf serum collected in Brazil. The results from virus neutralisation tests performed on sera collected from cattle in the herd of the infected calf, showed that these cattle had markedly higher antibody titres against the atypical pestivirus 'HoBi' than against Bovine Viral Diarrhoea Virus types 1 and 2, or Border Disease Virus. The results also supported, consequently, the results from the molecular analysis, and demonstrated that a 'HoBi'-like pestivirus had been introduced to, and was now circulating in the herd. This study is the first to report a natural infection in cattle with a virus related to this atypical pestivirus, and it suggests that this group of pestiviruses may already be spread in cattle populations. The findings have implications for BVD control and for the biosafety of vaccines and other biological products produced with foetal calf serum. Consequently, these atypical pestiviruses should be included in serological assays, and any diagnostic assay aimed at detection of pestiviruses in biological products or animals should be tested for its ability to detect them.

Nanobacteria-like particles: a threat to cell cultures

The main goal of this study is to alert researchers who work with cell cultures for the risk of contamination by structures called nanobacteria (NB). NB are tiny structures with size varying from 80 to 500 nm, commonly occurring in clusters and producing a biofilm which contains carbonate or hydroxyl apatite. The most likely source of cell culture contamination by such organisms is serum used as supplement in culture media. The presence of NB leads to a progressive culture deterioration with accumulation of granules (probably phagocytized NB) in cytoplasmic vacuoles, an increasing number of dead cells in the supernatant and degeneration of cells that remained attached to the bottom of the vessel. NB can also be found in culture supernatants where they are found in clusters with variable size and displaying brownian movement. In this study, 19 cell lineages, 8 batches of sera and 1 batch of growth supplement from different sources were analyzed. Samples from sera were cultured in Eagle’s Minimum Essential Medium (E-MEM) or incubated directly at 37oC. Tests carried out to detect the presence of extracellular bacteria, Mycoplasma sp and viruses were all negative. Analysis by scanning electron microscopy (SEM) revealed tiny oval structures less than 500 nm in size, isolated or in small groups, in all material analyzed except in one fetal bovine serum batch.

International Standard for serum vitamin B12 and serum folate: international collaborative study to evaluate a batch of lyophilised serum for B12 and folate content

Vitamin B(12) and folate measurements in serum show wide inter-methodology variability. This variability appears to be due in part to the lack of standardisation against internationally accepted reference materials. Pooled human serum, lyophilised in ampoules and designated 03/178, was therefore evaluated by 24 laboratories in seven countries for its suitability to serve as an International Standard (IS) for B(12) and folate. IS 03/178 was assayed using a range of commercial analysers, microbiological assays and, for folate, candidate reference methods based on liquid chromatography coupled to isotope-dilution tandem mass spectrometry (LC/MS/MS). Mean vitamin B(12) and folate values for reconstituted 03/178 across all laboratories and methods were 480 pg/mL [coefficient of variation (CV) 12.8%] and 5.52 ng/mL (CV 17.1%), respectively. The total folate content of reconstituted 03/178, determined using LC/MS/MS, was 12.1 nmol/L (equivalent to 5.33 ng/mL), made up of 9.75 nmol/L 5-methyl tetrahydrofolic acid (5MeTHF; CV 5.5%), 1.59 nmol/L 5-formyl tetrahydrofolic acid (5FoTHF; CV 4.2%) and 0.74 nmol/L folic acid (FA; CV 31.6%). The inclusion of three serum samples in the study with different B(12) and folate levels demonstrated a considerable reduction in inter-laboratory variability when the B(12) and folate content of the samples was determined relative to the IS 03/178 rather than to the analyser calibration. IS 03/178 demonstrated satisfactory long-term stability in accelerated degradation studies. Use of IS 03/178 to standardise serum B(12) and folate assays reduced inter-laboratory variability. The World Health Organization (WHO) Expert Committee on Biological Standardisation established 03/178 as the first IS for serum vitamin B(12) and serum folate, with assigned values of 480 pg/mL of vitamin B(12) and 12.1 nmol/L folate when the lyophilised contents of the ampoule are reconstituted with 1 mL of water.

Isolation of rat marrow-derived mesenchymal stem cells

Isolation of rat marrow-derived mesenchymal stem cells

Docosahexaenoic Acid Supports Cell Growth and Expression of Choline Acetyltransferase and Muscarinic Receptors in NG108-15 Cell Line

A large body of evidence indicates that adequate intake of polyunsaturated fatty acids is essential for brain development in early ontogenesis and positively impacts various pathological states connected with aging, as well as other neurodegenerative diseases (Jump, 2002; Bazan, 2003; Ruxton et al., 2004). In the present experiments, we investigated the possible effects of polyunsaturated docosahexanoic acid (DHA [22:6, n = 3]) on the expression of cholinergic phenotype-represented by choline acetyltransferase (ChAT) activity and a number of surface muscarinic receptors-as well as on cell growth in the cholinergic cell line NG108-15(Hamprecht, 1977; Hamprecht et al., 1985). However, chemical composition of different batches of sera is neither stable nor defined, and this fact complicates investigations on in vitro effects of substances that are natural constituents of serum. To avoid this restraint we employed defined medium in which fatty acid-free bovine albumin as a carrier of DHA replaced serum. Growth of most cell lines, as well as cells in primary cultures, depends strictly on the presence of serum in growth medium. As expected, withdrawal of serum resulted in growth arrest exemplified by a decrease in protein content compared with control cells grown in the presence of serum and also caused a decrease in ChAT activity (Fig. 1, lower left). DHA, at a concentration of 10 mumol/L, largely prevented both growth arrest in defined medium with fatty acid-free bovine albumin as a carrier of DHA and the attenuation of ChAT activity. DHA at concentrations 10 times higher had no further effect. At a concentration of 100 mumol/L, DHA also significantly increased the number of surface muscarinic receptors compared with cells grown in serum-containing as well as serum-free medium (Fig. 1, upper right). These data demonstrate the ability of DHA at low micromolar concentrations to support cell growth and expression of ChAT activity. Although it is not possible to stipulate a mechanism of action on the expression of ChAT and muscarinic receptors, a plausible explanation could be prevention of apoptosis, evidenced by a sharp decrease in executive caspase-3 activity (Fig. 1, lower right). Apoptosis is a process with a high requirement for energy. An improved metabolic state of cells consequent to suppression of apoptosis might thus better fulfill requirements for protein synthesis and targeting.

Germ layer induction from embryonic stem cells

Embryonic stem (ES) cells have the potential to develop into all cell types of the adult body. This capability provides the basis for considering the ES cell system as a novel and unlimited source of cells for replacement therapies for the treatment of a wide range of diseases. Before the cell-based therapy potential of ES cells can be realized, a better understanding of the pathways regulating lineage-specific differentiation is required. Current studies suggest that the bone morphogenic protein, transforming growth factor-beta, Wnt, and fibroblast growth factor pathways that are required for gastrulation and germ layer induction in the embryo are also essential for differentiation of ES cells in culture. The current understanding of how these factors influence germ layer induction in both the embryo and in the ES cell differentiation system is addressed in this review.

Detection and molecular characterisation of bovine polyomavirus in bovine sera in New Zealand

AIM: To investigate the prevalence of bovine polyomavirus (BPyV) DNA in commercial batches of bovine serum products, cell lines and cattle in New Zealand and to characterise the viral DNA detected. METHODS: Two nested polymerase chain reaction (PCR) assays were applied to detect BPyV in bovine sera. One was used to screen for the VP1 gene of BPyV DNA in: 140 batches of commercial bovine serum products, including 66 batches of fetal bovine serum (FBS), 34 batches of calf serum, and 40 batches of adult bovine serum (ABS)/plasma; 112 individual adult bovine sera; and 16 cell lines of various species origin. Fifty batches of serum samples were also tested, using the second nested PCR assay that screened for the Large T gene. Restriction fragment length polymorphism (RFLP) was conducted with 36 PCR products amplified from the VP1 gene of BPyV using EcoRI. Five selected VP1 PCR products were subjected to DNA sequencing and phylogenetic analysis. RESULTS: BPyV DNA was detected in 46 (70%) batches of FBS, 11 (32%) batches of calf sera and two (5%) batches of ABS/plasma, an overall prevalence of 42%. None of 112 adult bovine sera was BPyV-positive. RFLP analysis demonstrated a uniform digestion pattern in the majority (31/36) of amplicons tested, while the remaining PCR amplicons did not show enzyme cleavage. Sequence analysis of the PCR products (a 263 base pair (bp) fragment of the VP1 gene) obtained from five batches of FBS showed 96.2–98.9% homology to that of published sequences of BPyV. CONCLUSION: BPyV is a frequent contaminant of commercial bovine serum in New Zealand. The incidence of BPyV in adult bovine serum products is much lower than in FBS and calf serum. Genomic variations exist among different viruses. The clinical significance of the high prevalence of BPyV DNA in bovine serum products is yet to be determined.

Inhibition of lysyl oxidase activity can delay phenotypic modulation of chondrocytes in two-dimensional culture

Chondrocytes frequently de-differentiate in two-dimensional (2D) culture, especially in the presence of serum. To examine the role of lysyl oxidase (LOX) induced cross-linking in this phenomenon, the effect of the specific LOX inhibitor beta-aminopropionitrile (BAPN) was studied in 2D chondrocyte culture. Chick embryo sternal chondrocytes (both proliferative and hypertrophic, from caudal and cranial zones, respectively) were cultured in the presence and absence of BAPN. The production and activities of LOX and LOX-like (LOXL) were assessed by enzyme assay and the use of specific antibodies. Seventeen batches of serum of different origin were compared. Chondrocyte phenotype was assessed both morphologically and biochemically, the latter by quantitative analysis of production of radiolabeled cartilage collagens II, IX, X and XI, and the de-differentiation marker collagen I, for up to 4 weeks in culture. LOX and LOXL were identified, by Western blotting and immunofluorescence, and LO activity was measured in the medium, with both proliferative and hypertrophic chondrocytes. Inhibition of LO activity prevented or delayed chondrocyte de-differentiation, as characterized by changes in cell shape and synthesis of the five different collagen types, from the first days of culture for up to 4 weeks, depending on the origin of the serum added to the culture medium. LO activity may be involved in the control of chondrocyte phenotype, in addition to serum factors. Inhibition of LO activity by BAPN may be useful for the maintenance of the chondrocyte phenotype in 2D culture. Specific variations in the relative proportions of collagens II, IX and XI could be involved in the mechanism underlying these observations.

Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species

The genus Pestivirus within the family Flaviviridae currently consists of four different main species: Classical swine fever virus, Bovine viral diarrhea virus types 1 and 2 and Border disease virus. A fifth tentative species is represented by an isolate from a giraffe. In this study, a completely new pestivirus, isolated from a batch of fetal calf serum that was collected in Brazil, is described. It is proposed that the isolate D32/00_'HoBi' may constitute a novel sixth pestivirus species, because it is genetically, as well as antigenically, markedly different from all other pestiviruses. Based on the entire N(pro)- and E2-encoding sequences, identities of <70 % to all other pestivirus species were determined. Similarly, cross-neutralization and binding studies using antisera and mAbs revealed marked antigenic differences between D32/00_'HoBi' and all other pestiviruses.

An Immunogenic Non-Human Sialic Acid in Human ES Cells.

The potential of human embryonic stem cells (HESC) to differentiate into any cell type or tissue makes them excellent candidates for therapy of many diseases. To be safe for transplantation, HESC-derived cells and tissues must be free of xenogenic components that could trigger an immune response in the patient. Almost all currently existing HESC cell lines have been in contact with non-human cells (a mouse embryonic fibroblast feeder layer) and/or animal products (such as fetal calf serum or “serum replacements”). These xenogenic culture conditions not only increase the risk of infection by non-human pathogens but also, as we address here, the possibility for HESC to incorporate the immunogenic non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). We used flow cytometry to detect Neu5Gc on surfaces of HESC growing under such standard culture conditions and confirmed its presence by chemical analysis, showing that it represented up to 10% of the total sialic acids in HESC. In embryoid bodies (EB), the first step in HESC differentiation, the percentage of Neu5Gc ranged from 5% to 17%, despite being grown in the absence of the feeder layer and with a reduced concentration of serum. While the HESC could incorporate some Neu5Gc from the feeder layer, the major source appears to be the serum-replacement medium, which was found to be very rich in Neu5Gc (129 nmoles/mL). As many healthy humans have “natural” circulating anti-Neu5Gc antibodies (Tangvoranuntakul P et al. Proc Natl Acad Sci USA 2003; 100, 12045–12050), we asked if these antibodies could recognize the HESC growing under the standard conditions and if this binding caused activation of complement. Human IgG and complement factor C3b were indeed detected on HESC cell surfaces after exposure to human sera with high level of anti-Neu5Gc antibodies. Much less IgG or C3b was detected after exposure to human sera with low level such antibodies. To reduce the Neu5Gc content of HESC and EB, we substituted the serum-replacement medium with heat inactivated type AB pooled human serum batches that were also pretested to be very low in natural anti-Neu5Gc antibodies. HESC were able to maintain an undifferentiated state when cultured in such conditions, without losing the ability to differentiate into EB. After a week in the new medium, the percentage of Neu5Gc dropped to 1.2% in the HESC and to 0.2% in the EB. Moreover, this process markedly reduced the IgG and C3b deposition caused by exposure to human sera with high levels of anti-Neu5Gc antibodies. Thus, the metabolic uptake and incorporation of Neu5Gc by HESC growing under the currently accepted culture conditions could trigger an immune response against any HESC-derived transplant, if the recipient has naturally occurring anti-Neu5Gc antibodies. We demonstrate that growth and maintenance of HESC in human serum is feasible, and markedly reduces this risk. In practice, a short-term switch to serum from the transplant recipient should be sufficient to eliminate the Neu5Gc risk. And added advantage of this approach is that one can screen for any allogeneic cytotoxic antibodies that happen to be in the transplant recipient's serum.

The C4b-binding Protein-Protein S Complex Inhibits the Phagocytosis of Apoptotic Cells

The phagocytosis of apoptotic cells is a complex process involving numerous interactions between the target cell and the macrophage. We have examined a role of the major soluble inhibitor of the classic and lectin complement pathways, C4b-binding protein (C4BP), in the clearance of apoptotic cells. The major form of C4BP present in blood is composed of seven alpha-chains and one beta-chain, which binds protein S (PS). Approximately 70% of all PS in human plasma is trapped in such a complex and is able to localize C4BP to the surface of apoptotic cells due to the high affinity to phosphatidylserine. Free PS has recently been shown to enhance phagocytosis of apoptotic cells by macrophages. We observed a stimulatory effect of free PS on the engulfment of apoptotic cells (BL-41 and Jurkat) by primary human macrophages or THP-1 cells and a decrease of activity in serum depleted of PS in agreement with previous results. However, we also show that the process is strongly inhibited in the presence of the C4BP-PS complex. Addition of the C4BP-PS complex to serum deficient in both molecules abolished the enhancing effect of serum on phagocytosis. The effect of both free PS and the C4BP-PS complex could be inhibited with monoclonal antibody directed against the Gla domain of PS. Although the presence of the C4BP-PS complex on apoptotic cells may lead to decreased phagocytosis, it may still be beneficial to the host, since it could prevent secondary necrosis because it inhibits further complement attack.

Peripartum serum vitamin E, retinol, and beta-carotene in dairy cattle and their associations with disease.

Peripartum decreases in serum concentrations of vitamins A and E may contribute to impaired immune function in dairy cows. The objectives of this study were to describe peripartum serum concentrations of alpha-tocopherol, beta-carotene, and retinol and their associations with disease risk. On 20 farms over 1 yr, blood samples were collected weekly from 1057 cows from 1 wk before expected calving until 1 wk postpartum. Serum concentrations of alpha-tocopherol, beta-carotene, and retinol, as well as several biochemical variables were measured. Their associations with the risk of retained placenta or clinical mastitis were modeled separately with logistic regression, and the factors associated with the concentration of each vitamin were modelled with mixed linear regression. Differences in vitamin concentrations between 2 batches of sera analyzed 6 mo apart required stratification of statistical analyses. Accounting for the effects of parity, season, and twins, an increase in alpha-tocopherol of 1 microg/mL in the last week prepartum reduced the risk of retained placenta by 20%, whereas serum nonesterified fatty acid concentration > or = 0.5 mEq/L tended to increase risk of retained placenta by 80%. In the last week prepartum, a 100 ng/mL increase in serum retinol was associated with a 60% decrease in the risk of early lactation clinical mastitis. There were significant positive associations of peripartum serum concentrations among each of alpha-tocopherol, beta-carotene, and retinol.

Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.

In Vitro Differentiation of Mouse ES Cells: Bone and Cartilage

Publisher Summary The in vitro differentiation of ES cells, which closely recapitulates embryonic cell differentiation processes, has been used as a model system to analyze cell differentiation. This is an alternative way to investigate the consequences of loss of function mutations after gene targeting if knock-out mice cannot be generated. Because of their broad differentiation capacity ES cells are also discussed as an experimental approach to generate cells for transplantation. A common method to differentiate ES cells in vitro is their cultivation as cell aggregates, so-called “embryoid bodies (EBs).” The efficiency of ES cell-derived chondrocyte differentiation is found to be influenced by several parameters, such as the batch of fetal calf serum used for cultivation or the size of EBs. For a reproducible pattern of chondrogenic and osteogenic differentiation it is an important prerequisite to generate EBs of the same size. This is achieved by differentiation of ES cells via hanging drop-cultivation. This chapter describes some basic techniques of ES cell cultivation, the methods used for chondrogenic and osteogenic differentiation of ES cells, for characterization of the specific cell types and for isolation of chondrocytes from EBs. Furthermore, the chapter briefly summarizes approaches to enhance the differentiation efficiency.

Identification of vacuolar serine proteinase as a major allergen of Aspergillus fumigatus by immunoblotting and N-terminal amino acid sequence analysis.

Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning. The objective of this study is to identify A. fumigatus allergens through immunoblot analysis using sera from asthmatic patients. IgE-binding components of A. fumigatus and IgE cross-reactivity among allergens of different prevalent airborne fungal species were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens identified were determined by Edman degradation. Among two batches (70 and 41 sera) of asthmatic sera tested, 19 (27%) and 14 (34%), respectively, have IgE immunoblot reactivity towards components of A. fumigatus. A 34-kDa protein that reacts with IgE antibodies in 15 (79%) and 11 (79%) of the 19 and 14 positive samples, respectively, may be considered a major allergen of A. fumigatus. The N-terminal amino acid sequences of the 34 kDa major allergen and the 30.5 and 30 kDa IgE-binding components of A. fumigatus showed sequence identity to that of the vacuolar serine proteinase from A. fumigatus. The results from immunoblot inhibition show IgE cross-reactivity among major allergens of A. fumigatus, P. notatum and P. oxalicum. Results obtained suggest that the 34 kDa major allergen of A. fumigatus may be a vacuolar serine proteinase. There is IgE cross-reactivity among serine proteinase allergens of A. fumigatus, P. notatum and P. oxalicum.

Association of porous hydroxyapatite and bone marrow cells for bone regeneration

The preparation of hybrid material with osteoinductive capacity may be achieved by association of cultured autologous bone cells with a porous ceramic vehicle. We optimized culture conditions for rabbit marrow stromal stem cells (MSCs), notably by selection from batches of fetal calf serum. Rabbit MSCs formed colony-forming unit-fibroblastic (CFU-Fs) in vitro. Their alkaline phosphatase (ALP) activity was doubled in the presence of dexamethasone. Autologous rabbit serum allowed the formation of ALP-positive CFU-Fs, but results were highly variable depending on the rabbit. We tested the osteogenic potential of autologous cultured (with or without dexamethasone addition in the culture medium) and noncultured rabbit MSCs associated with a porous hydroxyapatite ceramic after a dorsal intramuscular implantation. Nucleated cells (10 7 or 10 8/mL) were used for the preparation of autologous hybrid material. A significantly higher number of implants containing bone was obtained with a suspension of 10 7 cells/mL cultured in the presence of 10 −8 M dexamethasone. Some positive implants were also obtained with a suspension of 10 8 noncultured cells/mL. We demonstrated the feasibility of preparing rabbit autologous hybrid materials following a process for controlling culture conditions, cell characterization and cell/material association.

Cell Culture Conditions Determine Apolipoprotein CIII Secretion and Regulation by Fibrates in Human Hepatoma HepG2 Cells

Fibrates are widely used drugs which lower triglycerides and increase HDL concentrations in serum. Recent findings from our laboratory have shown that fibrates repress apolipoprotein (apo) CIII gene expression, an effect that explains partially the triglyceride-lowering activity of these drugs. The goal of the present study was to compare the effect of various fibrates on apo CIII gene expression in the human hepatoblastoma cell line HepG2. First, we demonstrate that the level of apo CIII secretion by HepG2 cells is controlled by serum factors whereas apo CIII mRNA levels are not and even increase under conditions when apo CIII secretion dramatically decreases. Twelve different fetal calf serum batches were tested during this study and apo CIII secretion in cell medium could only be detected with three of them. The effect of serum on apolipoprotein secretion was more pronounced for apo CIII whereas other apolipoproteins (apo E, apo B, apo AII and apo AI) were affected to a lesser extent. Under serum conditions allowing apo CIII secretion, treatment with the peroxisome-proliferator activated receptor (PPAR)α activators fenofibrate, gemfibrozil and Wy-14643 result in a marked lowering of apo CIII secretion and gene expression, this effect being most pronounced with Wy-14643. Comparison of the activity of a PPARγ-specific ligand, the antidiabetic thiazolidinedione, BRL-49653 and a PPARα ligand Wy-14643 showed a marked decrease of apo CIII secretion and gene expression after activation of PPARα but not PPARγ. In conclusion, fibrates down-regulate apo CIII gene expression in human HepG2 cells, most likely via PPARα but not via PPARγ. However, these effects are only observed in HepG2 cells cultured under appropriate conditions.

Chimeric Human Epidermal Reconstructs to Study the Role of Melanocytes and Keratinocytes in Pigmentation and Photoprotection

Chimeric epidermal reconstructs made with Negroid melanocytes and Caucasoid keratinocytes (or vice versa) were studied before and after UVB irradiation to understand the respective roles of these cells in tanning and photoprotection, especially lipoperoxidation and enzymatic defences against free radicals. Using this approach, we have confirmed overall the theory of the epidermal melanin unit. We have also shown that melanocytes of poorly tanning Caucasoids, which have a comparatively higher content of unsaturated fatty acids in their cell membrane, are more prone to the peroxidative effects of UV light, and that keratinocytes participate in photoprotection via phototype-dependent antioxidant enzyme activities, especially for catalase.

Chronic granulomatous disease: Report on a registry of 368 patients

A gradual decrease in colony-forming ability of human fibroblast strains was found to be an early sign of cellular senescence. This decrease occurs in fibroblast strains from patients with ataxia-telangiectasia (A-T) at significantly earlier passage levels than with normal strains; accordingly, the lifespan of A-T cells in culture is shorter than that of normal strains. The colony-forming efficiency of A-T cells can serve as a quantitative measure for serum quality: A-T cells fail to form colonies, or have a markedly reduced colony-forming efficiency, in some batches of calf serum and newborn calf serum, whereas in other batches their clonal growth is maximal. Normal fibroblasts show the same colony-forming efficiency with all batches of serum. Fibroblast growth factor (FGF) and epidermal growth factor (EOF), but not insulin, were able to correct this deficiency in the growth-promoting ability of certain serum batches. In addition, conditioning medium containing these sera on confluent unirradiated or irradiated monolayers of any other human fibroblast strain had the same effect. A-T cells seem to have an increased demand for certain FGF- or EGF-like growth factors that are present in variable amounts in different serum batches and also in conditioned medium.

Control materials for selenium in serum suitable for speciation studies

Selenium is an essential element which importance for human health is widely recognised. At present, Se status is assessed by either determination of total Se or glutathione peroxidase activity, but increasing attention is paid to the dosage of other Se species. From an analytical point of view, the presence of different Se species may influence even the determination of total Se. Most analytical techniques suffer from interferences and require control (CMs) and reference (RMs) materials for their validation. At present, the preparation of materials with different Se concentrations mainly relies on the addition of inorganic Se compounds to human or animal serum. However, the analytical behaviour of added inorganic Se may be different from that of native organic Se species, thus affecting the evaluation of method and laboratory performances. CMs at different Se concentrations were prepared without the addition of inorganic Se by pooling batches of serum with low and high Se content obtained from two different animal species (cow and horse) and used for two years in the Italian External Quality Assessment Scheme (EQAS) for trace elements in biological fluids. The results obtained in this EQAS are reported and compared with those obtained by the same laboratories on CMs spiked with inorganic Se. CMs prepared according to this protocol could be suitable for the assessment of methods for the speciation of Se in serum.