An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.

We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.

The Impacts of Supplemental Protein during Development on Amino Acid Concentrations in the Uterus and Pregnancy Outcomes of Angus Heifers.

Replacement heifer development is one of the most critical components in beef production. The composition of the ideal uterine environment could maximize fertility and reproductive efficiency. Our hypothesis was that protein supplementation would affect the uterine environment of beef heifers without inhibiting development or reproduction. To test the effects of dietary supplementation on these outcomes, a randomized complete block design with repeated measures was implemented. Angus heifers (n = 60) were blocked by body weight (BW) and randomly assigned to one of three supplemental protein treatment groups (10% (CON), 20% (P20), and 40% (P40)). Mixed model ANOVAs were used to determine whether protein supplementation treatments, time, and the interaction or protein supplementation, semen exposure, and the interaction influenced uterine luminal fluid (ULF) and pregnancy outcomes. Amino acids (AAs) were impacted (p < 0.001), specifically, the essential AAs: Arg, Iso, Leu, Val, His, Lys, Met, Phe, Trp. Protein supplementation influenced multiple AAs post-insemination: Arg (p = 0.03), CC (p = 0.05), 1-MH (p = 0.001), and Orn (p = 0.03). In conclusion, protein supplementation did not affect the reproductive development via puberty attainment or the timing of conception even with alterations in growth. However, uterine AA concentrations did change throughout development and protein supplementation influenced ULF d 14 post-insemination, which may affect the conception rates.

The effects of differing nutritional levels and body condition score on scrotal circumference, motility, and morphology of bovine sperm.

Bulls often experience various levels of nutrient availability throughout the year. Nutritional management is a critical factor on overall ejaculate composition and the ability to get females pregnant. We hypothesized that differing nutritional levels and body condition score (BCS) affect reproductive fertility parameters in bulls. Mature Angus bulls (n = 11) were individually housed and randomly assigned to one of two dietary regimens: 1) over-fed (n = 5) or 2) restricted (n = 6). Bulls were fed the same ration at different volumes to achieve desired effects resulting in eight individual treatments: gain to an over-fed body condition score ([BCS]; GO), gain after nutrient restriction (GR), loss after an over-fed BCS (LO), loss from nutrient restriction (LR), maintenance at ideal adiposity (BCS = 6) after overfeeding (IMO), maintenance at ideal adiposity after nutrient restriction (IMR), maintenance at an over-fed BCS (BCS = 8; MO), and maintenance at a restricted BCS (BCS = 4; MR). Body weight (BW) and BCS were recorded every 2 wk to monitor bull weight and BCS changes. Scrotal circumference was measured every 28 d. Body fat and sperm motility and morphology were evaluated every 84 d. Scrotal circumference, motility, and morphology were normalized to the initial value of each bull. Thus, allowing the individual bull to serve as a control. Statistical analyses were conducted with PROC GLIMMIX of SAS as a complete randomized design to determine if treatment influenced BW, BCS, scrotal circumference, motility, morphology, and adipose thickness. Scrotal circumference (P < 0.001) had the least amount of deviation from initial during the LR (0.29 ±â€…0.44) treatment and the greatest during the MO (3.06 ±â€…0.44), LO (2.28 ±â€…0.44), MR (2.43 ±â€…0.44), GR (3.03 ±â€…0.44), and IMR (2.91 ±â€…0.44) treatments. Sperm motility was not affected by nutritional treatments (P = 0.55). Both head and total defects of sperm differed (P = 0.02) due to nutritional treatments. Increased head abnormalities occurred during the LO (37.60 ±â€…8.61) treatment, with no differences between the other treatments. Total defects increased during the LO (43.80 ±â€…9.55) treatment with similar increases in bulls during the GR (29.40 ±â€…9.55) and IMR (35.60 ±â€…9.55) treatments. In conclusion, male fertility was impacted when a deviation from a BCS of 6 occurred which could be detrimental to reproductive and beef production efficiency.

At least four distinct blue cationic phthalocyanine dyes sold as "alcian blue" raises the question: what is alcian blue?

We investigated physicochemical characteristics of dye lots sold as "alcian blue" using the Biological Stain Commission (BSC) precipitation test, differential scanning calorimetry, high performance liquid chromatography, thin layer chromatography and UV/visible spectroscopy. Four blue phthalocyanine dyes were detected in 11 commercial dye lots. These four included the original ingrain blue 1 CI 74000 dye and the dye sold with the name "alcian blue pyridine variant"; we discuss also the possible identity of the additional two dyes. A proposed extension to the BSC analytic scheme is presented that could distinguish three categories of commercial alcian blue dyes from each other and from the original alcian blue 8G.

The effects of protein level on cytokines and chemokines in the uterine environment of beef heifers during development.

The development of replacement heifers is crucial for breeding success and herd efficiency. Nutritional management can affect not only reproductive development but also the inflammatory status of the uterine environment, which may impact reproductive functions such as pregnancy establishment and development. The study herein evaluated the concentration of cytokines and chemokines in the uterus of heifers supplemented with different levels of protein. Angus heifers (n = 60) were blocked by body weight (BW) and randomly assigned to 1 of 3 treatments based on protein supplementation level: control of 10% crude protein (CON), 20% crude protein (P20), or 40% crude protein (P40). BW, body condition score, and blood samples were taken every 2 wk for 140 d to monitor development. Uterine flushes were performed monthly and concentrations of cytokines (IL-1α, IL-1ß, TNF-α, IFN-γ, IL-10, VEGF-α, IL-17A, and IL-36RA) and chemokines (IL-8, MCP-1, MIP-1α, and MIP-1ß) were quantified via ELISA multiplex. To test if there were mean differences in cytokines between the treatment groups or over time, PROC GLIMMIX (SAS v 9.4) was utilized. Concentrations of all cytokines and chemokines, except IL-1α, changed throughout heifer development (P < 0.05). Heifers in the P40 treatment group displayed reduced concentrations of MCP-1 (P = 0.007) and tended to have decreased concentrations of IFN-γ (P = 0.06). Cytokine IL-36RA tended (P = 0.06) to be affected by protein level, with the lowest concentrations observed in CON heifers. Most cytokines and chemokines increased following the initial month of supplementation (P < 0.05). The increase in concentrations after 1 mo may indicate an adaptive response in the uterus to diet change. Cytokines and chemokines fluctuated due to physiological changes occurring during development. Further research is needed to determine the influence of nutrition on uterine inflammation and long-term impacts on reproductive function.

Certification procedures used by the Biological Stain Commission for eriochrome cyanine R (C.I. 43820, Mordant blue 3).

Eriochrome cyanine R (C.I. 43820, Mordant blue 3), also known as chromoxane cyanine R and solochrome cyanine R, has been used as a biological stain since 1957. In conjunction with ferric ions, it provides selective blue coloration of the nuclei of cells in methods procedurally similar to commonly used progressive or regressive hemalum (aluminum-hematoxylin) stains. Eriochrome cyanine R also is used to stain the myelin sheaths of axons in nerve tissue; the results are visually similar to those in sections stained with luxol fast blue MBS (C.I. 74180, solvent blue 38) with selective blue coloration of myelin and erythrocytes. Eriochrome cyanine R is an article of commerce with many uses in industrial coloration and analytical chemistry; it can be used instead of either hematoxylin or luxol fast blue MBS, especially in the event of a shortage of either of the latter compounds. The Biological Stain Commission (BSC) will certify batches of eriochrome cyanine R that meet the criteria set out in this document. The criteria include satisfactory UV/visible spectra at pH 4 and pH 12 - 13, a dye content not less than 40% and not greater than 52% (calculated as the color acid; equivalent to 46 - 59% of the trisodium salt), and satisfactory performance in three staining methods: regressive for nuclei, progressive for nuclei and regressive for myelin.

Revised tests and standards for Biological Stain Commission certification of alcian blue dyes.

Alcian blue dyes are copper phthalocyanines with a variety of cationic side chains; they are useful for staining carbohydrate polyanions while avoiding staining of nucleic acids. The properties of the original alcian blue and of similar dyes with published chemical structures are reviewed here. Variation among samples submitted to the Biological Stain Commission (BSC) for certification has led to the recognition of two types of commercially available alcian blue at this time. The designation "alcian blue 8G or equivalent" is reserved for dyes that resemble alcian blue 8GX manufactured in the 1960s (CI 74240; ingrain blue 1). These dyes react with alkali to form an insoluble pigment that cannot be re-dissolved in acid. The name "alcian blue variant" is for similar dyes that do not form insoluble pigments; an alkali-induced precipitate, if formed, re-dissolves with acidification. For certification by the BSC, both types of alcian blue must dissolve in 3% acetic acid to make a 1% solution (pH close to 2.5), which must provide selective coloration of intestinal mucus, cartilage and mast cells, but not of nuclei. After alcian blue staining and treatment with 0.03 M Na2CO3 or Li2CO3 to convert the bound dye to a pigment, the Feulgen stain for DNA is applied. Dyes to be certified as alcian blue 8G or the equivalent must resist extraction by the 5 M HCl used in the Feulgen reaction. Dyes to be certified as alcian blue variant are not required to be convertible to acid-insoluble pigments, but they must dissolve easily in water at pH 5.7 containing 0.5 M magnesium chloride and the dye must remain in solution for at least 24 h. A critical electrolyte concentration (CEC) staining test also is described; this must be passed for certification of an alcian blue variant. Successful CEC staining is also a desirable property of alcian blue 8G or equivalent, but not essential for certification of an otherwise satisfactory batch. The spectrophotometric criteria for alcian blue dyes also are revised; a wider range of absorption maximum (605-634 nm) is allowed. The dye powders used in published staining techniques with the original alcian blue 8G were 40-60% dye, but some modern alcian blue dyes have dye content as high as 90%. The BSC's assay for dye content is not a criterion for certification, but it should influence the amount of dye to include in a staining solution.

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA, and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al3+ ions for each hematein molecule, at pH 2.0-2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular "background color." Such solutions contain a red cationic dye-metal complex and an excess of Al3+ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5-8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0-3.5. Staining by hemalum at pH 2.0-3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pKa ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al2(SO4)3 followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0-3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al3+ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al2(SO4)3 solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.

Preoperative HBA1c and risk of postoperative complications in patients with gynaecological cancer.

BACKGROUND: HBA1c is used as an indicator for the long-term control of the glycaemic state and outcome predictors in diabetic patients. Diabetic patients have an increased risk of post-operative complications especially those related to infection. The aim of our study is to ascertain the relationship between HBA1c levels and post-operative recovery within the subspecialty of gynaecological oncology. METHOD: Prospective cohort study during the period 1 August 2012 through 31 August 2014. Preoperative measurement of HBA1c on all gynaecological oncology patients that underwent major surgery. Patient variables collected and analysed were BMI (kg/m(2)), length of stay (LOS in days), cancer stage (stage 1 through stage 4), infective complications, non-infective complications and readmission to hospital. RESULTS: A total of 300 patients were included in our study, 34 of them were known to be diabetic while 266 were presumed to be non-diabetic. Of the presumed non-diabetic cohort, 17.3 % (46/266) had impaired glucose tolerance or diabetes. Mean BMI was significantly increased in the pre-existing diabetic group (32.8 vs. 29.3 kg/m(2), p = 0.016). Infective complications were almost double the rate amongst the known diabetic women than those presumed to be non-diabetic (32.4 vs. 18.0 %, p = 0.048). Rate of re-admission to hospital due to complications was 20.6 % in the diabetic group and 4.1 % within the presumed non-diabetic group (p < 0.001). Infective complications occurred in 16.9 % of women with HBA1c <42 mmol/mol, 22.7 % of those with HBA1c of 42-47 mmol/mol, 43.5 % of patients with HBA1c 48-64 mmol/mol and 37.5 % of patients with HBA1c >64 mmol/mol. Non-infective complications were also more frequent in women with elevated HBA1c (11.1, 22.7, 26.1 and 12.5 % in those women with HBA1c <42, 42-47, 48-64 and >64 mmol/mol, respectively). Re-admission to hospital within 30 days for a complication of surgery occurred in 4.4 % of women with HBA1c <42 mmol/mol, 4.5 % of women with HBA1c measured at 42-47 mmol/mol, 30.8 % of those with HBA1c 48-64 mmol/mol and 25 % of women with HBA1c >64 mmol/mol. CONCLUSION: Preoperative measurement of HBA1c may identify patients (both diabetic and non-diabetic women) at higher risk of postoperative complications and could be used as a trigger for modification of the perioperative management of such patients.

A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule.

The simple polychrome methylene blue (PMB) staining procedure for blood or tissue smears from dead animals (M'Fadyean reaction) established in 1903 remained accepted as a highly reliable, rapid diagnostic test for anthrax for six decades while that disease was still common in livestock throughout the world. Improvements in disease control led to anthrax becoming rare in industrialized countries and less frequent in developing countries with the result that quality controlled, commercially produced PMB became hard to obtain by the 1980s. Mixed results with alternative methylene blue-based stains then led to diagnosis failures, confusion among practitioners and mistrust of this procedure as a reliable test for anthrax. We now report that, for laboratories needing a reliable M'Fadyean stain at short notice, the best approach is to have available commercially pure azure B ready to constitute into a solution of 0.03 g azure B in 3 ml of 95% ethanol or methanol to which is then added 10 ml of 0.01% KOH (0.23% final azure B concentration) and which can then be used immediately and through to the end of the tests. Stored in the dark at room temperature, the shelf life is at least 12 months. Smears should be fixed with ethanol or methanol (95-100%), not by heat, and the stain left for 5 min before washing off for optimum effect.

Anatomy of the temporal lobe.

Only primates have temporal lobes, which are largest in man, accommodating 17% of the cerebral cortex and including areas with auditory, olfactory, vestibular, visual and linguistic functions. The hippocampal formation, on the medial side of the lobe, includes the parahippocampal gyrus, subiculum, hippocampus, dentate gyrus, and associated white matter, notably the fimbria, whose fibres continue into the fornix. The hippocampus is an inrolled gyrus that bulges into the temporal horn of the lateral ventricle. Association fibres connect all parts of the cerebral cortex with the parahippocampal gyrus and subiculum, which in turn project to the dentate gyrus. The largest efferent projection of the subiculum and hippocampus is through the fornix to the hypothalamus. The choroid fissure, alongside the fimbria, separates the temporal lobe from the optic tract, hypothalamus and midbrain. The amygdala comprises several nuclei on the medial aspect of the temporal lobe, mostly anterior the hippocampus and indenting the tip of the temporal horn. The amygdala receives input from the olfactory bulb and from association cortex for other modalities of sensation. Its major projections are to the septal area and prefrontal cortex, mediating emotional responses to sensory stimuli. The temporal lobe contains much subcortical white matter, with such named bundles as the anterior commissure, arcuate fasciculus, inferior longitudinal fasciculus and uncinate fasciculus, and Meyer's loop of the geniculocalcarine tract. This article also reviews arterial supply, venous drainage, and anatomical relations of the temporal lobe to adjacent intracranial and tympanic structures.

Euthanization methods influence cytokine mRNA expression levels in age 0 year Oncorhynchus mykiss.

Significant differences in cytokine transcription were found between Oncorhynchus mykiss euthanized using the pharmacological agents MS-222 v. benzocaine and also when contrasting death induced by carbon dioxide asphyxiation v. physical methods (cervical dislocation). This study highlights the need to consider the potentially confounding effect of euthanization method on gene expression data.

Certification procedures for sirius red F3B (CI 35780, Direct red 80).

Sirius red F3B (CI 35780, Direct red 80) is a polyazo dye used principally in staining methods for collagen and amyloid. For certification by the Biological Stain Commission, a sample of the dye must exhibit an absorption spectrum of characteristic shape with a maximum at 528-529 nm, a small shoulder near 500 nm and narrow peaks at 372, 281-282 and 230-235 nm. Spot tests (color changes with addition of concentrated H(2)SO(4) or HCl and subsequent dilution or neutralization) also are applied. The dye must perform satisfactorily in the picro-sirius red method for collagen by providing red staining of all types of collagen with yellow and green birefringence of fibers. Llewellyn's alkaline sirius red method applied to tissue known to contain amyloid must show red coloration of the products with green birefringence. Dye content, which does not influence significantly the staining properties of sirius red F3B, is not assayed.

Repeated oral administration of chitosan/DNA nanoparticles delivers functional FVIII with the absence of antibodies in hemophilia A mice.

BACKGROUND: Current treatment of hemophilia A is expensive and involves regular infusions of factor (F)VIII concentrates. The supply of functional FVIII is further compromised by the generation of neutralizing antibodies. Thus, the development of an alternative safe, cost effective, non-invasive treatment that circumvents immune response induction is desirable. OBJECTIVES: To evaluate the feasibility of oral administration of chitosan nanoparticles containing FVIII DNA to provide sustainable FVIII activity in hemophilia A mice. METHODS: Nanoparticles were characterized for morphology, DNA protection and transfection efficiency. Oral administration of nanoparticles containing canine FVIII in C57Bl/6 FVIII(-/-) hemophilia A mice was evaluated for biodistribution, plasma FVIII activity and phenotypic correction. Sustainable FVIII expression was elucidated after repeated nanoparticle administration. Immune responses to repeated oral nanoparticle administration were also investigated. RESULTS: Chitosan nanoparticles had a particle size range of 200-400 nm and protected DNA from endonuclease and pH degradation. In addition, nanoparticles transfected HEK 293 cells resulted in expression of eGFP, luciferase and FVIII. Hemophilia A mice that ingested chitosan nanoparticles demonstrated transient canine FVIII expression reaching > 100 mU 1 day after treatment, together with partial phenotypic correction. The delivered FVIII plasmid DNA was detected in the intestine and, to a lesser extent, in the liver. Importantly, repeated weekly administrations restored FVIII activity. Furthermore, inhibitors and non-neutralizing FVIII antibodies were not detectable. CONCLUSIONS: Repeat oral administration of FVIII DNA formulated in chitosan nanoparticles resulted in sustained FVIII activity in hemophilic mice, and thus may provide a non-invasive alternative treatment for hemophilia A.

Hematoxylin shortages: their causes and duration, and other dyes that can replace hemalum in routine hematoxylin and eosin staining.

The origins of repeated hematoxylin shortages are outlined. Lack of integration in the hematoxylin trade exacerbates the problems inherent in using a natural product. Separate corporations are engaged in tree growth and harvesting, dye extraction, processing of extracts to yield hematoxylin, and formulation and sale of hematoxylin staining solutions to the end users in biomedical laboratories. Hematoxylin has many uses in biological staining and no single dye can replace it for all applications. Probably, the most satisfactory substitutes for aluminum-hematoxylin (hemalum) are the ferric complexes of celestine blue (CI 51050; mordant blue 14) and eriochrome cyanine R (CI 43820; mordant blue 3, also known as chromoxane cyanine R and solochrome cyanine R). The iron-celestine blue complex is a cationic dye that binds to nucleic acids and other polyanions, such as those of cartilage matrix and mast cell granules. Complexes of iron with eriochrome cyanine R are anionic and give selective nuclear staining similar to that obtained with acidic hemalum solutions. Iron complexes of gallein (CI 45445; mordant violet 25), a hydroxyxanthene dye, can replace iron-hematoxylin in formulations for staining nuclei, myelin, and protozoa.

A system for quantitative evaluation of fixatives for light microscopy using paraffin sections of kidney and brain.

A numerical scoring system is presented for evaluating structural fixation of certain mammalian tissues for light microscopy. Small pieces of rat's kidney and brain, tissues for which artifacts of fixation are well documented, were fixed in various fluids. Random code numbers hid their identities, and paraffin sections were stained to show nuclear chromatin, cytoplasm and extracellular materials. Two sections of each specimen were examined and awarded scores according to described schemes, for microanatomical and cytological fixation. The assessment was confined to preservation of structure; chemical changes were not taken into account. When the code was broken, the scores for both sections of each specimen from each fixative were added. The scores obtained (24 best to eight worst) are generally comparable to the grades (I-V) given for traditional fixatives by JR Baker. The criteria for quality of fixation were defined more explicitly than those for Baker's grading method, which used mouse testis as the test object. Assessments are presented for several traditional fixatives and for four zinc-formaldehyde mixtures. The scoring system should be useful for evaluating newly developed fixatives for animal tissues for light microscopic examination of paraffin sections. In an evaluation of four traditional fixatives and four zinc-formaldehyde mixtures, variation among three different observers was only +/-1 point on either side of the median score for microanatomical or cytological preservation by any of the eight fixatives. This approach has certain limitations, notably that the criteria for cytological fixation do not include the preservation of chromosomes or specific cytoplasmic organelles.

Prognosis and treatment of spinal cord astrocytoma.

PURPOSE: To identify the prognostic factors for spinal cord astrocytoma and determine the effects of surgery and radiotherapy on outcome. METHODS AND MATERIALS: This retrospective study reviewed the cases of consecutive patients with spinal cord astrocytoma treated at Mayo Clinic Rochester between 1962 and 2005. RESULTS: A total of 136 consecutive patients were identified. Of these 136 patients, 69 had pilocytic and 67 had infiltrative astrocytoma. The median follow-up for living patients was 8.2 years (range, 0.08-37.6), and the median survival for deceased patients was 1.15 years (range, 0.01-39.9). The extent of surgery included incisional biopsy only (59%), subtotal resection (25%), and gross total resection (16%). Patients with pilocytic tumors survived significantly longer than those with infiltrative astrocytomas (median overall survival, 39.9 vs. 1.85 years; p < 0.001). Patients who underwent resection had a worse, although nonsignificant, median survival than those who underwent biopsy only (pilocytic, 18.1 vs. 39.9 years, p = 0.07; infiltrative, 19 vs. 30 months, p = 0.14). Postoperative radiotherapy, delivered in 75% of cases, gave no significant survival benefit for those with pilocytic tumors (39.9 vs. 18.1 years, p = 0.33) but did for those with infiltrative astrocytomas (24 vs. 3 months; Wilcoxon p = 0.006). On multivariate analysis, pilocytic histologic type, diagnosis after 1984, longer symptom duration, younger age, minimal surgical extent, and postoperative radiotherapy predicted better outcome. CONCLUSION: The results of our study have shown that histologic type is the most important prognostic variable affecting the outcome of spinal cord astrocytomas. Surgical resection was associated with shorter survival and thus remains an unproven treatment. Postoperative radiotherapy significantly improved survival for patients with infiltrative astrocytomas but not for those with pilocytic tumors.

News from the Biological Stain Commission.

In the three earlier editions of News from the Biological Stain Commission (BSC), under the heading of "Regulatory affairs," the BSC's International Affairs Committee reported on the work of Technical Committee 212, Clinical Laboratory Testing and in Vitro Diagnostic Test Systems of the International Standards Organization (ISO/TC 212) and its working groups, WG 1, WG 2 and WG 3. In this issue of News from the BSC, H.O. Lyon provides information from the annual meeting of ISO/TC 212 that took place June 2-4, 2008 in Vancouver, British Columbia, Canada. In addition, under the heading of "Certification," J.A. Kiernan examines the certification procedure for thionine used by the BSC laboratory in Rochester, NY.