Implementing human factors in anaesthesia: guidance for clinicians, departments and hospitals: Guidelines from the Difficult Airway Society and the Association of Anaesthetists: Guidelines from the Difficult Airway Society and the Association of Anaesthetists.

Human factors is an evidence-based scientific discipline used in safety critical industries to improve safety and worker well-being. The implementation of human factors strategies in anaesthesia has the potential to reduce the reliance on exceptional personal and team performance to provide safe and high-quality patient care. To encourage the adoption of human factors science in anaesthesia, the Difficult Airway Society and the Association of Anaesthetists established a Working Party, including anaesthetists and operating theatre team members with human factors expertise and/or interest, plus a human factors scientist, an industrial psychologist and an experimental psychologist/implementation scientist. A three-stage Delphi process was used to formulate a set of 12 recommendations: these are described using a 'hierarchy of controls' model and classified into design, barriers, mitigations and education and training strategies. Although most anaesthetic knowledge of human factors concerns non-technical skills, such as teamwork and communication, human factors is a broad-based scientific discipline with many other additional aspects that are just as important. Indeed, the human factors strategies most likely to have the greatest impact are those related to the design of safe working environments, equipment and systems. While our recommendations are primarily provided for anaesthetists and the teams they work with, there are likely to be lessons for others working in healthcare beyond the speciality of anaesthesia.

Human factors in anaesthesia: a narrative review.

Healthcare relies on high levels of human performance, as described by the 'human as the hero' concept. However, human performance varies and is recognised to fall in high-pressure situations, meaning that it is not a reliable method of ensuring safety. Other safety-critical industries embed human factors principles into all aspects of their organisations to improve safety and reduce reliance on exceptional human performance; there is potential to do the same in anaesthesia. Human factors is a broad-based scientific discipline which aims to make it as easy as possible for workers to do things correctly. The human factors strategies most likely to be effective are those which 'design out' the chance of an error or adverse event occurring. When errors or adverse events do happen, barriers are in place to trap them and reduce the risk of progression to patient and/or worker harm. If errors or adverse events are not trapped by these barriers, mitigations are in place to minimise the consequences. Non-technical skills form an important part of human factors barriers and mitigation strategies and include: situation awareness; decision-making; task management; and team working. Human factors principles are not a substitute for proper investment and appropriate staffing levels. Although applying human factors science has the potential to save money in the long term, its proper implementation may require investment before reward can be reaped. This narrative review describes what is known about human factors in anaesthesia to date.

Safety and efficacy of paramedic treatment of regular supraventricular tachycardia: a randomised controlled trial.

INTRODUCTION: Supraventricular tachycardias (SVTs) are a common cause of acute hospital presentations. Adenosine is an effective treatment. To date, no studies have directly compared paramedic-with hospital-delivered treatment of acute SVT with adenosine. METHOD: Randomised controlled trial comparing the treatment of SVT and discharge by paramedics with conventional emergency department (ED)-based care. Patients were excluded if they had structural heart disease or contraindication to adenosine. Discharge time, follow-up management, costs and patient satisfaction were compared. RESULTS: Eighty-six patients were enrolled: 44 were randomised to paramedic-delivered adenosine (PARA) and 42 to conventional care (ED). Of the 37 patients in the PARA group given adenosine, the tachycardia was successfully terminated in 81%. There was a 98% correlation between the paramedics' ECG diagnosis and that of two electrophysiologists. No patients had any documented adverse events in either group. The discharge time was lower in the PARA group than in the ED group (125 min (range 55-9513) vs 222 min (range 72-26 153); p=0.01), and this treatment strategy was more cost-effective (£282 vs £423; p=0.01). The majority of patients preferred this management approach. Being treated and discharged by paramedics did not result in the patients being less likely to receive ongoing management of their arrhythmia and cardiology follow-up. CONCLUSIONS: Patients with SVT can effectively and safely be treated with adenosine delivered by trained paramedics. Implementation of paramedic-delivered acute SVT care has the potential to reduce healthcare costs without compromising patient care. TRIAL REGISTRATION NUMBER: NCT02216240.

A forward modelling approach for the estimation of oxygen extraction fraction by calibrated fMRI.

The measurement of the absolute rate of cerebral metabolic oxygen consumption (CMRO2) is likely to offer a valuable biomarker in many brain diseases and could prove to be important in our understanding of neural function. As such there is significant interest in developing robust MRI techniques that can quantify CMRO2 non-invasively. One potential MRI method for the measurement of CMRO2 is via the combination of fMRI and cerebral blood flow (CBF) data acquired during periods of hypercapnic and hyperoxic challenges. This method is based on the combination of two, previously independent, signal calibration techniques. As such analysis of the data has been approached in a stepwise manner, feeding the results of one calibration experiment into the next. Analysing the data in this manner can result in unstable estimates of the output parameter (CMRO2), due to the propagation of errors along the analysis pipeline. Here we present a forward modelling approach that estimates all the model parameters in a one-step solution. The method is implemented using a regularized non-linear least squares approach to provide a robust and computationally efficient solution. The proposed framework is compared with previous analytical approaches using modelling studies and in vivo acquisitions in healthy volunteers (n=10). The stability of parameter estimates is demonstrated to be superior to previous methods (both in vivo and in simulation). In vivo estimates made with the proposed framework also show better agreement with expected physiological variation, demonstrating a strong negative correlation between baseline CBF and oxygen extraction fraction. It is anticipated that the proposed analysis framework will increase the reliability of absolute CMRO2 measurements made with calibrated BOLD.

Renal cell carcinoma containing macroscopic fat on CT mimics an angiomyolipoma due to bone metaplasia without macroscopic calcification.

We report a case of renal cell carcinoma (RCC) containing foci of macroscopic fat, which were pathologically proven to be areas of osseous metaplasia. The macroscopic fat was not associated with calcification on the pre-operative CT scan. To our knowledge, there are no reported cases of RCC that contain osseous metaplasia without evidence of macroscopic calcification on CT. The finding is significant because standard imaging practice is to classify a renal mass containing intratumoral macroscopic fat that is not associated with calcification, ossification or invasion of perirenal or hilar fat as an angiomyolipoma.

Long-term efficacy of catheter ablation for atrial fibrillation: impact of additional targeting of fractionated electrograms.

OBJECTIVES: To investigate long-term efficacy of catheter ablation for atrial fibrillation (AF) and the impact of ablating complex or fractionated electrograms (CFEs) in addition to pulmonary vein isolation and linear lesions in persistent AF (PeAF). METHODS: Consecutive cases from 2002-2007 were analysed. All the patients underwent a wide-area circumferential ablation with confirmation of electrical isolation. For PeAF, linear lesions were added, with additional targeting of CFE from 2005. Data were collected in a prospective database. Attempts were made to contact all patients for follow-up. RESULTS: 285 patients underwent 530 procedures. The mean (SD) age was 57 (11) years, 75% were male, 20% had structural heart disease and 53% had paroxysmal AF (PAF). The mean number of procedures was 1.9 per patient (1.7 for PAF and 2.0 for PeAF). Procedural complications included stroke or transient ischemic attack in 0.6% and pericardial effusion requiring drainage in 1.7%. During 2.7 years (0.2 to 7.4 years) of follow-up from the last procedure, there were seven deaths (unrelated to their ablation or AF) and three strokes or transient ischemic attack (0.3% per year). Freedom from AF/atrial tachyarrhythmia was 86% for PAF and 68% for PeAF. Late recurrence was 3 per 100 years of follow-up after >3 years. The Kaplan-Meier analysis showed that CFE ablation improved the outcome for PeAF after the first cluster of procedures (p=0.049), with a trend towards improved final outcome (p=0.130). CONCLUSIONS: Long-term freedom from AF is achievable in most patients with PAF and PeAF with low rates of late recurrence. Additional targeting of CFE improves outcome for PeAF. Late adverse events including stroke are few.

Neurocognitive contributions to verbal fluency deficits in frontotemporal lobar degeneration.

OBJECTIVE: To test the hypothesis that different neurocognitive networks underlie verbal fluency deficits in frontotemporal lobar degeneration (FTLD). METHODS: Letter ("FAS") and semantic ("animal") fluency tests were administered to patients with a behavioral/dysexecutive disorder (bvFTLD; n = 71), semantic dementia (SemD; n = 21), and progressive nonfluent aphasia (PNFA; n = 26). Tests measuring working memory, naming/lexical retrieval, and semantic knowledge were also obtained. MRI voxel-based morphometry (VBM) studies were obtained on a subset of these patients (bvFTLD, n = 51; PNFA, n = 11; SemD, n = 10). RESULTS: Patients with SemD were disproportionately impaired on the semantic fluency measure. Reduced output on this test was correlated with impaired performance on naming/lexical retrieval tests. VBM analyses related reduced letter and semantic fluency to anterior and inferior left temporal lobe atrophy. Patients with bvFTLD were equally impaired on both fluency tests. Poor performance on both fluency tests was correlated with low scores on working memory and naming/lexical retrieval measures. In this group, MRI-VBM analyses related letter fluency to bilateral frontal atrophy and semantic fluency to left frontal/temporal atrophy. Patients with PNFA were also equally impaired on fluency tests. Reduced semantic fluency output was correlated with reduced performance on naming/lexical retrieval tests. MRI-VBM analyses related semantic fluency to the right frontal lobe and letter fluency to left temporal atrophy. CONCLUSIONS: Distinct neurocognitive networks underlie impaired performance on letter and semantic fluency tests in frontotemporal lobar degeneration subgroups.

Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro.

Recent studies indicate that a rare population of primitive quiescent BCR-ABL(+) cells are innately insensitive to imatinib mesylate (IM) and persist after IM therapy of patients with chronic myeloid leukemia (CML). New approaches to the eradication of these cells are therefore likely to be crucial to the development of curative therapies for CML. We have now found that Ara-C, LY294002 (a PI-3 (phosphatidylinositol-3' kinase) kinase inhibitor), 17AAG (a heat-shock protein (HSP)-90 antagonist) and lonafarnib (a farnesyltransfease inhibitor) all enhance the toxicity of IM on K562 cells and on the total CD34(+) leukemic cell population from chronic phase CML patients. However, for quiescent CD34(+) leukemic cells, this was achieved only by concomitant exposure of the cells to lonafarnib. Ara-C or LY294002 alone blocked the proliferation of these cells but did not kill them, and Ara-C, LY294002 or 17AAG in combination with IM enhanced the cytostatic effect of IM but did not prevent the subsequent regrowth of the surviving leukemic cells. These studies demonstrate the importance of in vitro testing of novel agents on the subset of primary leukemic cells most likely to determine long-term treatment outcomes in vivo.

The effect of interstate travel on sleep patterns of elite Australian Rules footballers.

The purpose of the current study was to examine the effect of interstate air travel on the quality and quantity of sleep in elite Australian Rules football players. Ten elite male athletes, who were members of a Western Australian-based Australian Football League (AFL) team, participated in the study. Sleep pattern was assessed by measuring sleep duration (SLD), sleep efficiency (SE), number of wakings (NW) and total wake time after sleep onset (WT) using a wrist-worn actigraph. Subjective sleep quality (SQ) was assessed using a scale of sleep rating. Throughout the 2002 AFL season, measurements were obtained on the night before (N1), the night of (N2) and the night after (N3) home and away games. Baseline measurements were obtained from five consecutive non-game nights. Compared to baseline, SLD on N1 was increased when home and away (by 51 and 105 mins respectively, p<0.05), while all other measures of sleep pattern were unchanged. On N2, SLD was decreased to a similar degree whether home or away (by 68 and 64 mins respectively, p<0.05), while all other measures of sleep pattern were unchanged. By N3 all measures of sleep pattern had returned to baseline values. Relative to baseline, perception of SQ was worst on N2 of a home game. This study has shown that interstate travel by elite AFL players has no adverse effects on sleep pattern on the night before a game.

CML leukapheresis products can be enriched for CD34+ cells and simultaneously depleted of CD15+ cells using a simple Ab cocktail.

BACKGROUND: CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells. METHODS: Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. RESULTS: The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained. DISCUSSION: A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.

Intraspecific competition in populations of Helix aspersa with different histories of exposure to lead.

A range of invertebrates have become adapted to certain toxic metals, such that, in the presence of the contaminant, some measure of their performance, typically growth or reproduction, is superior to that of an unexposed population. Under such a toxic stress, a population with a history of exposure might outperform a naïve population in competition for limited resources. This study compared the shell growth of laboratory-bred juveniles from six populations of Helix aspersa with different histories of exposure to Pb. In 10 trials using various combinations of two populations, the snails competed for a limited supply of food that contained either no Pb or 500 micrograms/g-1 Pb, for 98 days. Each trial consisted of 10 juveniles, five from each population and was replicated four times. Nearly all of the food provided was consumed quickly after presentation. The total amount of shell growth within each replicate (the sum of the mean growth of the two populations) was highly consistent between trials so that the total amount of shell built was limited by food availability. The presence of Pb in the diet caused no measurable depression of shell growth and exposure history did not appear to confer any competitive advantage or disadvantage in any of the trials. One population consistently grew faster than its competitors in every trial of which it was a part. Shell growth tended to be greater in smaller juveniles. Snail activity is known to be inhibited at high densities and this may have contributed to the lower incremental growth in individuals kept at the higher densities. The competitive advantage enjoyed by one population may be primarily determined by their activity or perhaps their Ca metabolism.

Gender differences in carbohydrate loading are related to energy intake.

We demonstrated that female endurance athletes did not increase their muscle glycogen concentration after an increase in the dietary carbohydrate intake (58 --> 74%), whereas men did (Tarnopolsky MA, SA Atkinson, SM Phillips, and JD McDougall, J Appl Physiol 78: 1360-1368, 1995). This may have been related to a lower energy or carbohydrate intake by the women or due to an inherent gender difference in glycogen storage capacity. We examined whether well-trained men (n = 6) and women (n = 6) increased muscle glycogen concentration after an increase in both the relative (58 --> 75%) and absolute energy and carbohydrate intake and whether potential gender differences were related to muscle hexokinase enzyme activity. Subjects were randomly allocated to three diets [Hab, habitual; CHO, high carbohydrate (75%); and CHO + E, extra energy + CHO ( upward arrow~34%)] for a 4-day period before a muscle biopsy for analysis of total and pro- and macroglycogen and hexokinase activity. Total glycogen concentration was higher for the men on the CHO and CHO + E trials compared with Hab (P < 0.05), whereas women increased only on the CHO + E trial compared with Hab (P < 0.05). There were no gender differences in the proportion of pro- and macroglycogen or hexokinase activity. A low energy intake may explain the previously reported lower capacity for women to glycogen load compared with men.

Development, characterization and therapy of a disseminated model of childhood neuroblastoma in SCID mice.

PURPOSE: To develop a highly reproducible model of disseminated childhood neuroblastoma in mice to allow secondary evaluation of therapeutics against microscopic disseminated disease. METHODS: CB17/Icr SCID were injected i.v. with 10(3) to 5 x 10(6) human NB-1691 neuroblastoma cells. NB-1691 cells were detected by PCR for synaptophysin and tyrosine hydroxylase in peripheral blood, and bone marrow. Therapeutic studies evaluated topotecan and vincristine as single agents or in combination. Topotecan was administered i.v. daily for 5 days on two consecutive weeks. Courses were repeated every 21 days for three cycles. Vincristine (1 mg/kg) was administered i.v. every 7 days for nine consecutive weeks. Treatment started 11-21 days after tumor cell inoculation. RESULTS: Following injection of > or = 1 x 10(5) cells 100% of mice developed disease. Mice inoculated with 10(7) cells survived a median of 42 days. Survival time was a linear function of the cell inoculum. At autopsy, gross tumor was routinely detected in many organs in particular liver, ovaries, kidneys and adrenals. NB-1691 cells were detected by PCR in peripheral blood, and bone marrow. Immunohistochemical staining showed that lesions were strongly positive for synaptophysin, chromogranin A and negative for leukocyte common antigen. Topotecan (0.6 mg/kg) alone extended median survival from 44 days (controls) to 95 days. When treatment was started 21 days after inoculation of NB-1691 cells, topotecan extended median survival from 39 days (controls) to 91 and 99 days at dose levels of 0.3 and 0.6 mg/kg, respectively. Vincristine (1 mg/kg) extended survival by a median of 9.5 days. In combination with vincristine (1 mg/kg), median survival was increased to 141 days (topotecan 0.6 mg/kg) and 159 days (topotecan 1.0 mg/kg). CONCLUSION: This model of disseminated neuroblastoma is highly reproducible. As this model may more closely simulate childhood disease it may be a valuable adjunct in developing new approaches to advanced stage, poor prognosis neuroblastoma.

Relation between 9-aminocamptothecin systemic exposure and tumor response in human solid tumor xenografts.

9-Aminocamptothecin (9-AC) is a topoisomerase I inhibitor with activity against xenografts from childhood solid tumors; however, clinical trials with this compound have been disappointing, resulting in discontinuation of further development. The objectives of this study were to evaluate the antitumor activity of 9-AC in a panel of pediatric solid tumor xenografts and to relate the 9-AC lactone systemic exposure, defined as area under the concentration time curve (AUC), to the antitumor dose associated with tumor regression in the xenograft model. We evaluated protracted administration of i.v. and oral therapies (daily times 5) for 1, 2, or 3 weeks and for 1 or 3 cycles. The minimum effective dose of 9-AC causing objective regression of advanced tumors was determined for each schedule. 9-AC lactone plasma concentration-time profiles associated with the lowest dose achieving complete and partial responses for each xenograft were then determined for each regimen. Tumors were highly sensitive to 9-AC therapy, but the systemic exposure required for antitumor effect is in excess of that achievable in patients.

Serving street-dwelling individuals with psychiatric disabilities: outcomes of a psychiatric rehabilitation clinical trial.

OBJECTIVES: This study tested a psychiatric rehabilitation approach for organizing and delivering services to street-dwelling persons with severe mental illness. METHODS: Street-dwelling persons with severe mental illness were randomly assigned to the experimental program (called Choices) or to standard treatment in New York City. We assessed study participants at baseline and at 6-month intervals over 24 months, using measures of service use, quality of life, health, mental health, and social psychological status. The average deviation from baseline summary statistic was employed to assess change. RESULTS: Compared with persons in standard treatment (n = 77), members of the experimental group (n = 91) were more likely to attend a day program (53% vs 27%), had less difficulty in meeting their basic needs, spent less time on the streets (55% vs 28% reduction), and spent more time in community housing (21% vs 9% increase). They showed greater improvement in life satisfaction and experienced a greater reduction in psychiatric symptoms. CONCLUSIONS: With an appropriate service model, it is possible to engage disaffiliated populations, expand their use of human services, and improve their housing conditions, quality of life, and mental health status.

Antitumor activity of temozolomide combined with irinotecan is partly independent of O6-methylguanine-DNA methyltransferase and mismatch repair phenotypes in xenograft models.

The activity of temozolomide combined with irinotecan (CPT-11) was evaluated against eight independent xenografts (four neuroblastomas, three rhabdomyosarcomas, and one glioblastoma). In all studies, temozolomide was administered p.o. daily for 5 consecutive days/cycle, found in preliminary studies to be the optimal schedule for administration. Irinotecan was administered i.v. for 5 days for 2 consecutive weeks/cycle. Treatment cycles were repeated every 21 days for a total of three cycles over 8 weeks. In combination, temozolomide and CPT-11 induced complete responses in four neuroblastomas, two rhabdomyosarcomas, and the glioblastoma line. The activity of the combination was significantly greater than the activity of either agent administered alone in four tumor lines. Of interest, the interaction appeared independent of tumor MGMT or mismatch repair phenotype, suggesting that the mechanism of synergy may be independent of O6-methylation by temozolomide. Pharmacokinetic studies indicated no detectable interaction between these two agents. Further, coadministration of CPT-11 appeared to reduce the toxicity of temozolomide in tumor-bearing mice.

Synergy of topotecan in combination with vincristine for treatment of pediatric solid tumor xenografts.

Topotecan and vincristine were evaluated alone or in combination against 13 independent xenografts and 1 vincristine-resistant derivative, representing childhood neuroblastoma (n = 6), rhabdomyosarcoma (n = 5), or brain tumors (n = 3). Topotecan was given by i.v. bolus on a schedule found previously to be optimal. Drug was administered daily for 5 days on 2 consecutive weeks with cycles repeated every 21 days over a period of 8 weeks. Doses of topotecan ranged from 0.16 to 1.5 mg/kg to simulate clinically achievable topotecan lactone plasma systemic exposures. Vincristine was administered i.v. every 7 days at a fixed dose of 1 mg/kg. Given as a single agent, vincristine induced complete responses (CRs) in all mice bearing two rhabdomyosarcomas (Rh28 and Rh30) and some CRs in Rh12-bearing mice (57%) but relatively few CRs (<29%) in other tumors. As a single agent, topotecan induced CR in a low proportion of tumor lines. A dose-response model with a logit link function was used to investigate whether the combination of topotecan and vincristine resulted in greater than expected responses compared with the activity of the agents when administered alone. Only CR was used to evaluate tumor responses. The combination resulted in significantly greater than expected CRs than individual agents in nine tumor lines (four neuroblastoma, three brain tumors, and two rhabdomyosarcomas). Similar event-free (failure) distributions were shown in SJ-GBM2 glioblastoma xenografts, whether vincristine was administered on day 1 or day 5 of each topotecan course. To determine whether the increased antitumor activity with the combination was attributable to a change in drug disposition, extensive pharmacokinetic studies were performed. However, little or no interaction between these two agents was determined. Toxicity of the combination was marked by prolonged thrombocytopenia and decreased hemoglobin. However, approximately 75 and 80% of the maximum tolerated dose of each single agent, topotecan (1.5 mg/kg) or vincristine (1 mg/kg), could be given in combination, resulting in a combination toxicity index of approximately 1.5. These results show that the therapeutic effect of combining topotecan with vincristine was greater than additive in most tumor models of childhood solid tumors, and toxicity data suggest that this can be administered to mice with only moderate reduction in the dose levels for each agent.

Comparison of activation of CPT-11 by rabbit and human carboxylesterases for use in enzyme/prodrug therapy.

Several recent studies have examined the possibility of producing tumor-specific cytotoxicity with various enzyme/ prodrug combinations. The enzymes are targeted to tumor cells either with antibodies (ADEPT, antibody directed enzyme prodrug therapy) or with viruses (VDEPT). The goal of the present study was to identify an appropriate enzyme for use in activating the prodrug 7-ethyl-10-[4-(1-piper-idino)-1-piperidino]carbonyloxycamptothe cin (CPT-11). In this study, we compared the efficiency of CPT-11 metabolism by rabbit and human carboxylesterases in in vitro and in situ assays. Although the rabbit and human enzymes are very similar (81% identical; 86% homologous) and the active site amino acids are 100% identical, the rabbit enzyme was 100-1000-fold more efficient at converting CPT-11 to SN-38 in vitro and was 12-55-fold more efficient in sensitizing transfected cells to CPT-11. In vivo, Rh30 rhabdomyosarcoma cells expressing the rabbit carboxylesterase and grown as xenografts in immune-deprived mice were also more sensitive to CPT-11 than were control xenografts or xenografts expressing the human enzyme. Each of the three types of xenografts regressed when the mice were treated with CPT-11 given i.v. at 2.5 mg of CPT-11/kg/daily for 5 days/week for 2 weeks [(dx5)2] (one cycle of therapy), repeated every 21 days for a total of three cycles. However, following cessation of treatment, recurrent tumors were detected in seven of seven mice bearing control Rh30 xenografts and in two of seven mice bearing Rh30 xenografts that expressed the human enzyme. No tumors recurred in mice bearing xenografts that expressed the rabbit carboxylesterase. We conclude that rabbit carboxylesterase/CPT-11 may be a useful enzyme/prodrug combination.

Selection of CD34+ cells from cryopreserved PBPC can be significantly improved by the addition of recombinant human DNase (Pulmozyme).

BACKGROUND: It has been reported previously that PBPC can be recovered from cryopreservation and can be efficiently CD34-selected, to provide a product of high purity (> 80% CD34) with good yield (> 50% recovery). METHODS: In this study, we have investigated the effects of thawing and CD34-selecting cryopreserved PBPC in the presence of recombinant human deoxyribonuclease (rhDNase; Pulmozyme) and magnesium chloride (MgCl2 injection). RESULTS: The addition of Pulmozyme and MgCl2 significantly improves the yield of CD34+ cells, compared with the standard procedure (65.2% and 39.7%, respectively). Following CD34 selection, significantly greater recovery of CFC in the selected fraction can be obtained from Pulmozyme-treated cells, compared with standard cells. The use of recombinant human Pulmozyme and i.v. grade MgCl2 should facilitate the application of this procedure to the clinical setting. CD34+ cells selected from cryopreserved PBPC, can in turn be cryopreserved for a second time. When thawed, these cells still retained good viability (> 80%). DISCUSSION: Cells originally processed in the presence of Pulmozyme gave significantly superior yields of CD34+ cells and CFC compared with standard cells. The functional ability of these CD34+ cells was demonstrated further in an ex vivo expansion culture system with extensive proliferation of cells and CFC. In addition, the presence of significant numbers of primitive hemopoietic cells could be readily demonstrated in a cobblestone-area forming assay.