Impact of Preanalytical Factors During Histology Processing on Section Suitability for Digital Image Analysis.

Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables-staining protocol design, reagent quality, section attributes, and instrumentation-on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.

Methods Optimization for Routine Sciatic Nerve Processing in General Toxicity Studies.

Recent "best practice" recommendations for peripheral nervous system sampling and processing provide guidance regarding nerve preparation for animal toxicity studies. This study explored the impact of delayed fixation, type of fixative, processing cycle times, starting ethanol concentration, and water bath temperature to improve nerve preservation in routinely prepared (paraffin-embedded, hematoxylin and eosin [H&E]-stained) sections. Sciatic nerves from adult Wistar rats (diameter, 1.04 ± 0.1 mm) and young domestic pigs (diameter 5.9 ± 1.2 mm) fixed at necropsy ("0" hours) or 3, 6, 12, or 24 hours after death were immersed in neutral-buffered 10% formalin containing 1.2% methanol (NBF) or methanol-free 4% formaldehyde (MFF) at room temperature. After fixation for 24 hours (rat) or 48 hours (pig), specimens were processed into paraffin, and ∼5-µm-thick sections were flattened on water baths set at 35°C, 40°C, or 45°C before H&E staining. Large-diameter nerves (pig) required longer processing cycles to ensure sufficient paraffin infiltration. For both small-diameter (rat) and large-diameter nerves, structural integrity was optimal if fixation by NBF or MFF occurred within 3 hours and the initial ethanol concentration for tissue processing was lowered to 50%. At all time points, structural preservation of nerve fibers was acceptable using NBF but was better with MFF. Use of a water bath at 35°C reduced processing-related nerve fiber separation within sections.

Scientific and Regulatory Policy Committee (SRPC) paper: validation of digital pathology systems in the regulated nonclinical environment.

Digital Pathology Systems (DPS) are dynamic, image-based computer systems that enable the acquisition, management, and interpretation of pathology information generated from digitized glass slides. This article provides a roadmap for (1) qualification of a whole slide scanner (WSS) during a validation project, (2) validation of software required to generate the whole slide image (WSI), and (3) an introduction to visual digital image evaluation and image analysis. It describes a validation approach that can be utilized when validating a DPS. It is not the intent of this article to provide guidance on when validation of DPS is required. Rather, the article focuses on technical aspects of validation of the WSS system (WSS, IT infrastructure, and associated software) portion of a DPS and covers the processes of setting up the WSS for scanning a glass slide through saving a WSI on a server. Validation of a computerized system, such as a DPS, for use in a regulated nonclinical environment is governed by Code of Federal Regulations (CFR) Title 21 part 11: Electronic Records; Electronic Signature and predicate rules associated with Good Laboratory Practices documents including 21 CFR part 58. Similar regulation and predicate rules apply in the European Union and Japan.

Guide for collecting and reporting metadata on protocol variables and parameters from slide-based histotechnology assays to enhance reproducibility.

The central tenet of scientific research is the rigorous application of the scientific method to experimental design, analysis, interpretation, and reporting of results. In order to confer validity to a hypothesis, experimental details must be transparent and results must be reproducible. Failure to achieve this minimum indicates a deficiency in rationale, design, and/or execution, necessitating further experimental refinement or hypothesis reformulation. More importantly, rigorous application of the scientific method advances scientific knowledge by enabling others to identify weaknesses or gaps that can be exploited by new ideas or technology that inevitably extend, improve, or refine a hypothesis. Experimental details, described in manuscript materials and methods, are the principal vehicle used to communicate procedures, techniques, and resources necessary for experimental reproducibility. Recent examination of the biomedical literature has shown that many published articles lack sufficiently detailed methodological information to reproduce experiments. There are few broadly established practice guidelines and quality assurance standards in basic biomedical research. The current paper provides a framework of best practices to address the lack of reporting of detailed materials and methods that is pervasive in histological slide-based assays. Our goal is to establish a structured framework that highlights the key factors necessary for thorough collection of metadata and reporting of slide-based assays.

Validating Whole Slide Imaging Systems for Diagnostic Purposes in Pathology.

CONTEXT.­: The original guideline, "Validating Whole Slide Imaging for Diagnostic Purposes in Pathology," was published in 2013 and included 12 guideline statements. The College of American Pathologists convened an expert panel to update the guideline following standards established by the National Academies of Medicine for developing trustworthy clinical practice guidelines. OBJECTIVE.­: To assess evidence published since the release of the original guideline and provide updated recommendations for validating whole slide imaging (WSI) systems used for diagnostic purposes. DESIGN.­: An expert panel performed a systematic review of the literature. Frozen sections, anatomic pathology specimens (biopsies, curettings, and resections), and hematopathology cases were included. Cytology cases were excluded. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, the panel reassessed and updated the original guideline recommendations. RESULTS.­: Three strong recommendations and 9 good practice statements are offered to assist laboratories with validating WSI digital pathology systems. CONCLUSIONS.­: Systematic review of literature following release of the 2013 guideline reaffirms the use of a validation set of at least 60 cases, establishing intraobserver diagnostic concordance between WSI and glass slides and the use of a 2-week washout period between modalities. Although all discordances between WSI and glass slide diagnoses discovered during validation need to be reconciled, laboratories should be particularly concerned if their overall WSI-glass slide concordance is less than 95%.

Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats.

Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

Optical density-based image analysis method for the evaluation of hematoxylin and eosin staining precision.

Staining quality and reproducibility are essential factors to monitor laboratory quality assurance. In the last decade, there has been an increase in the use of digital pathology and image analysis. While the adoption of these tools provides a potential means to track staining precision by optical density (OD), it also presents challenges. Results from image analysis are more sensitive to variations in staining than microscopic evaluation by a pathologist. There are two goals with this study. The first was to track the precision of hematoxylin and eosin (H&E) staining, in both nuclear and cytoplasmic components by OD. The second was to determine the impact of different pre-analytical and analytical variables on the OD results. Specifically, the endpoints investigated were quality parameters including impacts of section thickness, protocol manipulation, expired hematoxylin on staining precision and reproducibility of staining over time. Our results show that image analysis of H&E-stained tissue sections is a viable tool for assessing and verifying staining quality. We also show that OD analysis results for H&E-stained sections are affected by changing pre-analytical and/or reagent variables. These authors chose a graphical rather than fully statistical analysis of the results to highlight the utility of visual aids in demonstrating H&E staining reproducibility.

An Image Analysis Solution For Quantification and Determination of Immunohistochemistry Staining Reproducibility.

With immunohistochemical (IHC) staining increasingly being used to guide clinical decisions, variability in staining quality and reproducibility are becoming essential factors in generating diagnoses using IHC tissue preparations. The current study tested a method to track and quantify the interrun, intrarun, and intersite variability of IHC staining intensity. Our hypothesis was that staining precision between laboratory sites, staining runs, and individual slides may be verified quantitatively, efficiently and effectively utilizing algorithm-based, automated image analysis. To investigate this premise, we tested the consistency of IHC staining in 40 routinely processed (formalin-fixed, paraffin-embedded) human tissues using 10 common antibiomarker antibodies on 2 Dako Omnis instruments at 2 locations (Carpinteria, CA: 30 m above sea level and Longmont, CO: 1500 m above sea level) programmed with identical, default settings and sample pretreatments. Digital images of IHC-labeled sections produced by a whole slide scanner were analyzed by a simple commercially available algorithm and compared with a board-certified veterinary pathologist's semiquantitative scoring of staining intensity. The image analysis output correlated well with pathology scores but had increased sensitivity for discriminating subtle variations and providing reproducible digital quantification across sites as well as within and among staining runs at the same site. Taken together, our data indicate that digital image analysis offers an objective and quantifiable means of verifying IHC staining parameters as a part of laboratory quality assurance systems.

Clade F AAVHSCs cross the blood brain barrier and transduce the central nervous system in addition to peripheral tissues following intravenous administration in nonhuman primates.

The biodistribution of AAVHSC7, AAVHSC15, and AAVHSC17 following systemic delivery was assessed in cynomolgus macaques (Macaca fascicularis). Animals received a single intravenous (IV) injection of a self-complementary AAVHSC-enhanced green fluorescent protein (eGFP) vector and tissues were harvested at two weeks post-dose for anti-eGFP immunohistochemistry and vector genome analyses. IV delivery of AAVHSC vectors produced widespread distribution of eGFP staining in glial cells throughout the central nervous system, with the highest levels seen in the pons and lateral geniculate nuclei (LGN). eGFP-positive neurons were also observed throughout the central and peripheral nervous systems for all three AAVHSC vectors including brain, spinal cord, and dorsal root ganglia (DRG) with staining evident in neuronal cell bodies, axons and dendritic arborizations. Co-labeling of sections from brain, spinal cord, and DRG with anti-eGFP antibodies and cell-specific markers confirmed eGFP-staining in neurons and glia, including protoplasmic and fibrous astrocytes and oligodendrocytes. For all capsids tested, 50 to 70% of glial cells (S100-ß+) and on average 8% of neurons (NeuroTrace+) in the LGN were positive for eGFP expression. In the DRG, 45 to 62% of neurons and 8 to 12% of satellite cells were eGFP-positive for the capsids tested. eGFP staining was also observed in peripheral tissues with abundant staining in hepatocytes, skeletal- and cardio-myocytes and in acinar cells of the pancreas. Biodistribution of AAVHSC vector genomes in the central and peripheral organs generally correlated with eGFP staining and were highest in the liver for all AAVHSC vectors tested. These data demonstrate that AAVHSCs have broad tissue tropism and cross the blood-nerve and blood-brain-barriers following systemic delivery in nonhuman primates, making them suitable gene editing or gene transfer vectors for therapeutic application in human genetic diseases.

National Society for Histotechnology and Digital Pathology Association Online Self-Paced Digital Pathology Certificate of Completion Program.

The field of digital pathology has rapidly expanded within the last few years with increasing adoption and growth in popularity. As digital pathology matures, it is apparent that we need well-trained individuals to manage our whole-slide imaging systems. This editorial introduces the joint National Society for Histotechnology and Digital Pathology Association online self-paced digital pathology certificate program which was launched in May 2018 that was established to meet this demand. An overview of how this program was developed, the content of the educational modules, and the way that this program is being offered is discussed.

Quantitative Image Analysis of Human Epidermal Growth Factor Receptor 2 Immunohistochemistry for Breast Cancer: Guideline From the College of American Pathologists.

CONTEXT.­: Advancements in genomic, computing, and imaging technology have spurred new opportunities to use quantitative image analysis (QIA) for diagnostic testing. OBJECTIVE.­: To develop evidence-based recommendations to improve accuracy, precision, and reproducibility in the interpretation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) for breast cancer where QIA is used. DESIGN.­: The College of American Pathologists (CAP) convened a panel of pathologists, histotechnologists, and computer scientists with expertise in image analysis, immunohistochemistry, quality management, and breast pathology to develop recommendations for QIA of HER2 IHC in breast cancer. A systematic review of the literature was conducted to address 5 key questions. Final recommendations were derived from strength of evidence, open comment feedback, expert panel consensus, and advisory panel review. RESULTS.­: Eleven recommendations were drafted: 7 based on CAP laboratory accreditation requirements and 4 based on expert consensus opinions. A 3-week open comment period received 180 comments from more than 150 participants. CONCLUSIONS.­: To improve accurate, precise, and reproducible interpretation of HER2 IHC results for breast cancer, QIA and procedures must be validated before implementation, followed by regular maintenance and ongoing evaluation of quality control and quality assurance. HER2 QIA performance, interpretation, and reporting should be supervised by pathologists with expertise in QIA.

Increased expression of host iron-binding proteins precedes iron accumulation and calcification of primary lung lesions in experimental tuberculosis in the guinea pig.

The growth and virulence of Mycobacterium tuberculosis depends on its ability to scavenge host iron, an essential and limited micronutrient in vivo. In this study, we show that ferric iron accumulates both intra- and extra-cellularly in the primary lung lesions of guinea pigs aerosol-infected with the H37Rv strain of M. tuberculosis. Iron accumulated within macrophages at the periphery of the primary granulomatous lesions while extra-cellular ferric iron was concentrated in areas of lesion necrosis. Accumulation of iron within primary lesions was preceded by an increase in expression of heavy chain (H) ferritin, lactoferrin and receptors for transferrin, primarily by macrophages and granulocytes. The increased expression of intra-cellular H ferritin and extra-cellular lactoferrin, more so than transferrin receptor, paralleled the development of necrosis within primary lesions. The deposition of extra-cellular ferric iron within necrotic foci coincided with the accumulation of calcium and phosphorus and other cations in the form of dystrophic calcification. Primary lung lesions from guinea pigs vaccinated with Mycobactrium bovis BCG prior to experimental infection, had reduced iron accumulation as well as H ferritin, lactoferrin and transferrin receptor expression. The amelioration of primary lesion necrosis and dystrophic calcification by BCG vaccination was coincident with the lack of extra-cellular ferric iron and lactoferrin accumulation. These data demonstrate that BCG vaccination ameliorates primary lesion necrosis, dystrophic mineralization and iron accumulation, in part by down-regulating the expression of macrophage H ferritin, lactoferrin and transferrin receptors, in vivo.

A long-acting, mono-PEGylated human growth hormone analog is a potent stimulator of weight gain and bone growth in hypophysectomized rats.

Recombinant human GH is used to treat GH deficiency in children and adults and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5-kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity, and in vivo potency. In vitro bioactivity of GH was reduced 100- to 1000-fold by extensive amine PEGylation of the protein. Here we describe a homogeneously modified, mono-PEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life, and increased potency in vivo. The mono-PEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20-kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH after sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable with that produced by daily administration of GH in hypophysectomized rats. Long-acting, mono-PEGylated GH analogs such as PEG-T3C are promising candidates for future testing in humans.

Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.

OBJECTIVE: Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the pegylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. MATERIALS AND METHODS: Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monopegylated derivatives of 12 cysteine analogs were prepared using 20-kDa maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild-type Epo (BV Epo) and a pegylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats. RESULTS: Biological activities of 17 purified Epo cysteine analogs and 10 purified pegylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only pegylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The pegylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation. CONCLUSIONS: Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monopegylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed Epo proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific pegylation appears to improve the pharmacokinetic properties of Epo.

Rapamycin modulation of p70 S6 kinase signaling inhibits Rift Valley fever virus pathogenesis.

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.

Enhanced circulating half-life and hematopoietic properties of a human granulocyte colony-stimulating factor/immunoglobulin fusion protein.

OBJECTIVE: The aim of this study was to determine whether fusion proteins comprising human granulocyte colony-stimulating factor (G-CSF) joined to human immunoglobulin G1 and G4 (IgG1 and IgG4) Fc and C(H) domains are biologically active and have improved pharmacokinetic and hematopoietic properties in vivo. MATERIAL AND METHODS: Chimeric genes encoding human G-CSF fused to the N-termini of the Fc and C(H) domains of human IgG1 and IgG4 were constructed and used to transfect monkey COS cells. The fusion proteins were purified from the conditioned media by protein A affinity chromatography. Bioactivities of the proteins were measured in a G-CSF-dependent in vitro bioassay. Pharmacokinetic and granulopoietic properties of the G-CSF/IgG1-Fc fusion protein were measured in normal rats. RESULTS: The G-CSF/IgG-Fc and G-CSF/IgG-C(H) fusion proteins were secreted from transfected COS cells primarily as disulfide-linked homodimers. On a molar basis, the purified G-CSF/IgG-Fc fusion proteins were as active as G-CSF in in vitro bioassays, whereas bioactivities of the purified G-CSF/IgG-C(H) fusion proteins were decreased 3- to 4-fold. The G-CSF/IgG1-Fc fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating neutrophils and white blood cells than G-CSF following intravenous and subcutaneous administration to rats. CONCLUSION: Fusion of G-CSF to human IgG domains results in homodimeric fusion proteins possessing high in vitro bioactivities, long circulating half-lives, and enhanced hematopoietic properties in vivo.

Hematopoietic properties of granulocyte colony-stimulating factor/immunoglobulin (G-CSF/IgG-Fc) fusion proteins in normal and neutropenic rodents.

Previously we showed that granulocyte colony-stimulating factor (G-CSF) in vitro bioactivity is preserved when the protein is joined via a flexible 7 amino acid linker to an immunoglobulin-1 (IgG1)-Fc domain and that the G-CSF/IgG1-Fc fusion protein possessed a longer circulating half-life and improved hematopoietic properties compared to G-CSF in normal rats. We have extended this analysis by comparing the relative hematopoietic potencies of G-CSF/IgG1-Fc to G-CSF in normal mice and to G-CSF and polyethylene glycol (PEG) -modified G-CSF in neutropenic rats. Mice were treated for 5 days using different doses and dosing regimens of G-CSF/IgG1-Fc or G-CSF and circulating neutrophil levels in the animals measured on Day 6. G-CSF/IgG1-Fc stimulated greater increases in blood neutrophils than comparable doses of G-CSF when administered using daily, every other day or every third day dosing regimens. In rats made neutropenic with cyclophosphamide, G-CSF/IgG1-Fc accelerated recovery of blood neutrophils to normal levels (from Day 9 to Day 5) when administered as 5 daily injections or as a single injection on Day 1. By contrast, G-CSF accelerated neutrophil recovery when administered as 5 daily injections, but not when administered as a single injection. G-CSF/IgG1-Fc was as effective as PEG-G-CSF at accelerating neutrophil recovery following a single injection in neutropenic rats. G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins in which the 7 amino acid linker was deleted also were effective at accelerating neutrophil recovery following a single injection in neutropenic rats. These studies confirm the enhanced in vivo hematopoietic properties of G-CSF/IgG-Fc fusion proteins.

PEGylation improves the pharmacokinetic properties and ability of interferon gamma to inhibit growth of a human tumor xenograft in athymic mice.

Interferon gamma (IFN-γ) is a 28 kDa homodimeric cytokine that exhibits potent immunomodulatory, anti-proliferative, and antiviral properties. The protein is used to treat chronic granulomatous disease and malignant osteopetrosis, and it is under investigation as a treatment for a variety of cancer, fungal and viral diseases. IFN-γ has a short circulating half life in vivo, which necessitates frequent administration to patients. An unusual feature of IFN-γ is that the protein contains no native cysteines. To create a longer-acting and potentially more effective form of the protein, we introduced a cysteine residue into the IFN-γ coding sequence at amino acid position 103, which is located in a surface-exposed, non-helical region of the protein. The added cysteine residue served as the site for targeted modification of the protein with a cysteine-reactive polyethylene glycol (PEG) reagent. The recombinant protein was expressed in bacteria, purified and modified with 10, 20, and 40 kDa maleimide PEGs. The purified, PEGylated proteins had in vitro bioactivities comparable to IFN-γ, as measured using an in vitro cell growth inhibition assay. The PEGylated proteins displayed 20- to 32-fold longer half lives than IFN-γ in rats, and they were significantly more effective than IFN-γ at inhibiting growth of a human tumor xenograft in athymic mice.