Calibration errors of Goldmann tonometers in a tertiary eye care centre.

BACKGROUND: To evaluate the calibration errors of all Goldmann applanation tonometers used clinically in the Vancouver General Hospital/University of British Columbia (VGH/UBC) Eye Care Centre. METHODS: All Goldmann tonometers used for patient care in the VGH/UBC Eye Care Centre were individually assessed for calibration errors. The tonometers were checked according to the Haag-Streit method using a standard calibration weight bar at 3 pressure levels (0, 20, 60 mm Hg). Calibration errors were classified into 3 categories: +/-0.5 mm Hg, +/-1.0 mm Hg, or +/-1.5 mm Hg or more. Ophthalmologists using tonometers with calibration errors > +/-1.0 mm Hg were notified, with the suggestion that the tonometers be returned to the manufacturer for recalibration. RESULTS: A total of 25 tonometers were evaluated. At the 20 mm Hg level, 17 (68%) fell within the manufacturer's recommended calibration range of +/-0.5 mm Hg, and 2 (8%) had errors of +/-1.0 mm Hg, resulting in 19 tonometers (76%) with errors of +/-1.0 mm Hg. Six tonometers (24%) were identified as having calibration errors greater than +/-1.0 mm Hg. INTERPRETATION: Calibration error of +/-1.0 mm Hg or less is clinically acceptable. Seventy-six percent of the Goldmann applanation tonometers in the Eye Care Centre were in the acceptable calibration range. As the number of acceptably calibrated tonometers was high, despite the infrequency of recent calibration, we feel that monthly evaluation of Goldmann tonometers is suitable.

Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables.

Retroviral transduction of human hematopoietic stem cells is still limited by lack of information about conditions that will maximize stem cell self-renewal divisions in vitro. To address this, we first compared the kinetics of entry into division of single human CD34+CD38- cord blood (CB) cells exposed in vitro to three different flt3-ligand (FL)-containing cytokine combinations. Of the three combinations tested, FL + hyperinterleukin 6 (HIL-6) yielded the least clones and these developed at a slow rate. With either FL + Steel factor (SF) + HIL-6 + thrombopoietin (TPO) or FL + SF + interleukin 3 (IL-3) + IL-6 + granulocyte-colony-stimulating factor (G-CSF), >90% of the cells that formed clones within 6 days undertook their first division within 4 days, although not until after 24 hours. These latter two, more stimulatory, cytokine combinations then were used to assess the effect of duration of cytokine exposure on the efficiency of transducing primitive CB cells with a gibbon ape leukemia virus-pseudotyped murine retroviral vector containing the enhanced green fluorescent protein (GFP) cDNA and the neomycin resistance gene. Fresh lin- CB cells exposed once to medium containing this virus plus cytokines on fibronectin-coated dishes yielded 23% GFP+ CD34+ cells and 52-57% G418-resistant CFC when assessed after 2 days. Prestimulation of the target cells (before exposing them to virus) with either the four or five cytokine combination increased their susceptibility. In both cases, the effect of prestimulation assessed using the same infection protocol was maximal with 2 days of prestimulation and resulted in 47-54% GFP+ CD34+ cells and 67-69% G418-resistant CFC. Repeated daily addition of new virus (up to three times), with assessment of the cells 2 days after the last addition of fresh virus, gave only a marginal improvement in the proportion of transduced CD34+ cells and CFC, but greatly increased the proportion of transduced LTC-IC (from 40% to >99%). Transplantation of lin- CB cells transduced using this latter 6-day protocol into NOD/SCID mice yielded readily detectable GFP+ cells in 10 of 11 mice that were engrafted with human cells. The proportion of the regenerated human cells that were GFP+ ranged from 0.2-72% in individual mice and included both human lymphoid and myeloid cells in all cases. High-level reconstitution with transduced human cells was confirmed by Southern blot analysis. These findings demonstrate that transplantable hematopoietic stem cells in human CB can be reproducibly transduced at high efficiency using a 6-day period of culture in a retrovirus-containing medium with either FL + SF + HIL-6 + TPO or FL + SF + IL-3 + IL-6 + G-CSF in which virus is added on the third, fourth, and fifth day.

High-efficiency retroviral transduction of mammalian cells on positively charged surfaces.

The efficiency of retroviruses as transducing agents has been appreciated for many years, particularly for hematopoietic cell targets for which alternative strategies applicable to adherent cells are not effective. Advances in vector design, pseudotyping, and infection conditions have eliminated the need to cocultivate the target cells with virus-producing cells. Nevertheless, improvements are still needed for many applications, including those with a therapeutic or clinical cell-tracking objective. In this study we show that more positively charged surfaces, including those designed for the culture of anchorage-dependent cells, allow measurable levels of adhesion by different pseudotypes of retroviruses, which can result in increased gene transfer efficiencies to a variety of target cells including normal primary human hematopoietic cells as well as human leukemic cell lines and rat and murine fibroblasts. In the experiments with primary human cells, equal aliquots of enriched CD34+ cord blood cells were first stimulated for 2 days with cytokines (Flt3 ligand, Steel factor, IL-3, IL-6, and G-CSF) and then exposed for 4 days to a green fluorescent protein (GFP)- and Neo(r)-encoding retrovirus produced in PG13 cells. Both the final yield (approximately 300% relative to initial numbers), and the proportion (approximately 60%) of transduced CD34+ cells, colony-forming cells, and long-term culture-initiating cells were the same for cells infected either in tissue culture dishes or in fibronectin-coated petri dishes. Similar proportions (approximately 10%) and absolute yields of GFP+ human cells were also found in multilineage engrafted NOD/SCID mice assessed 6 to 8 weeks after being transplanted with these two types of transduced, but unselected, cells. These findings suggest a new and simpler approach for achieving high gene transfer efficiencies to hematopoietic cells.

Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin.

The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34(+)CD38(-) cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neo(r) virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% +/- 2% GFP(+) CD34(+)CD38(-) cells, 75% +/- 10% G418-resistant clonogenic progenitors, and 50% +/- 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3x) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-beta(2)M(-/-)) mice were also efficiently transduced (10%-20% GFP(+) human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.