Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use.

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.

Isolation of early hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell population of cryopreserved, banked UC blood.

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors, whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed, washed, immunomagnetically depleted of cells expressing glycophorin A and CD14, reacted for flow cytometric detection of ALDH, and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H, 10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed, banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid, myeloid and megakaryocytic blood elements.

Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy.

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br), and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations), to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD, n=30) nucleated cells and was enriched in cells expressing CD34, CD117, CD105, CD127, CD133 and CD166, and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells, and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes, osteoblasts and chondrocytes. DISCUSSION: Hematopoietic, endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications.

Safety of hemodynamic augmentation in patients treated with Guglielmi detachable coils after acute aneurysmal subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Guglielmi detachable coils (GDC) used in the treatment of intracranial aneurysms do not always completely occlude the aneurysm. Thus, after an acute subarachnoid hemorrhage (SAH), there is a theoretical risk of rebleeding from coiled aneurysms, especially when blood pressure is elevated. The aim of this study is to determine whether use of hemodynamic augmentation (HA) to treat delayed ischemic deficits (DID) will increase the risk of rebleeding in these patients. METHODS: Delayed ischemic deficits developed in 12 (7 women and 5 men, aged 31 to 64 years) of 51 patients treated with GDC for acute SAH over a 4-year period. Aneurysms in all 12 patients were >/=80% obliterated with GDC, and there was >/=90% obliteration of 78% of the aneurysms. Hemodynamic augmentation with fluids, phenylephrine, dopamine, and/or dobutamine was used to treat DID for a mean duration of 3 days (range 1 to 11 days). RESULTS: With HA, mean arterial blood pressure (MAP) rose 15% (range 0 to 30%) and systolic blood pressure (SBP) rose 13% (range 0 to 29%) above baseline. MAP was maintained at >10% above baseline for 65% of the treatment period. The maximum MAP was 104 to 170 mm Hg (mean 140 mm Hg), and maximum SBP was 154 to 261 mm Hg (mean 210 mm Hg). No patient had rebleeding or any significant complication during the course of therapy. CONCLUSIONS: Based on this limited series of patients, we believe that it may be safe to use HA in patients treated with GDC for SAH.

Hemodynamic monitoring in the neurological intensive care unit.

The neurointensivist needs to have a thorough understanding of hemodynamic issues and the interaction of the brain and the cardiovascular system. Before one decides to intervene and try to correct an apparent "abnormal hemodynamic parameter" one needs to think whether such an intervention is indeed warranted and what effect the intervention would have on the cerebral circulation. The neurointensivist thus needs to approach these issues differently from the approach an internist or general intensivist would take.

Reversible left ventricular dysfunction associated with raised troponin I after subarachnoid haemorrhage does not preclude successful heart transplantation.

Transient abnormalities in ECGs, echocardiograms, and cardiac enzymes have been described in the acute setting of subarachnoid haemorrhage. In addition, left ventricular dysfunction has been reported at the time of brain death. A patient with an acute subarachnoid haemorrhage who presented with raised troponin I (TnI) concentrations and diffuse left ventricular dysfunction is described. After declaration of brain death 32 hours later, the heart was felt initially not suitable for transplantation. A normal cardiac catheterisation, however, lead to successful transplantation of the donor heart. Raised catecholamine concentrations and metabolic perturbations have been proposed as the mechanisms leading to the cardiac dysfunction seen with brain death. This may be a biphasic process, allowing time for myocardial recovery and reversal of the left ventricular dysfunction. Awareness of this phenomenon in the acutely ill neurologic population needs to be raised in order to prevent the unnecessary rejection of donor hearts.

No reduction in cerebral metabolism as a result of early moderate hyperventilation following severe traumatic brain injury.

OBJECT: Hyperventilation has been used for many years in the management of patients with traumatic brain injury (TBI). Concern has been raised that hyperventilation could lead to cerebral ischemia; these concerns have been magnified by reports of reduced cerebral blood flow (CBF) early after severe TBI. The authors tested the hypothesis that moderate hyperventilation induced early after TBI would not produce a reduction in CBF severe enough to cause cerebral energy failure (CBF that is insufficient to meet metabolic needs). METHODS: Nine patients were studied a mean of 11.2+/-1.6 hours (range 8-14 hours) after TBI occurred. The patients' mean Glasgow Coma Scale score was 5.6+/-1.8 and their mean age 27+/-9 years; eight of the patients were male. Intracranial pressure (ICP), mean arterial blood pressure, and jugular venous oxygen content were monitored and cerebral perfusion pressure was maintained at a level higher than 70 mm Hg by using vasopressors when needed. Measurements of CBF, cerebral blood volume (CBV), cerebral metabolic rate for oxygen (CMRO2), oxygen extraction fraction (OEF), and cerebral venous oxygen content (CvO2) were made before and after 30 minutes of hyperventilation to a PaCO2 of 30+/-2 mm Hg. Ten age-matched healthy volunteers were used as normocapnic controls. Global CBF, CBV, and CvO2 did not differ between the two groups, but in the TBI patients CMRO2 and OEF were reduced (1.59+/-0.44 ml/100 g/minute [p < 0.01] and 0.31+/-0.06 [p < 0.0001], respectively). During hyperventilation, global CBF decreased to 25.5+/-8.7 ml/100 g/minute (p < 0.0009), CBV fell to 2.8+/-0.56 ml/100 g (p < 0.001), OEF rose to 0.45+/-0.13 (p < 0.02), and CvO2 fell to 8.3+/-3 vol% (p < 0.02); CMRO2 remained unchanged. CONCLUSIONS: The authors conclude that early, brief, moderate hyperventilation does not impair global cerebral metabolism in patients with severe TBI and, thus, is unlikely to cause further neurological injury. Additional studies are needed to assess focal changes, the effects of more severe hyperventilation, and the effects of hyperventilation in the setting of increased ICP.

Neural pathways in tactile object recognition.

OBJECTIVE: To define further the brain regions involved in tactile object recognition using functional MRI (fMRI) techniques. BACKGROUND: The neural substrates involved in tactile object recognition (TOR) have not been elucidated. Studies of nonhuman primates and humans suggest that basic motor and somatosensory mechanisms are involved at a peripheral level; however, the mechanisms of higher order object recognition have not been determined. METHODS: The authors investigated 11 normal volunteers utilizing fMRI techniques in an attempt to determine the neural pathways involved in TOR. Each individual performed a behavioral paradigm with the activated condition involving identification of objects by touch, with identification of rough/smooth as the control. RESULTS: Data suggest that in a majority of individuals, TOR involves the calcarine and extrastriatal cortex, inferior parietal lobule, inferior frontal gyrus, and superior frontal gyrus-polar region. CONCLUSIONS: TOR may utilize visual systems to access an internal object representation. The parietal cortices and inferior frontal regions may be involved in a concomitant lexical strategy of naming the object being examined. Frontal polar activation likely serves a role in visuospatial working memory or in recognizing unusual representations of objects. Overall, these findings suggest that TOR could involve a network of cortical regions subserving somatosensory, motor, visual, and, at times, lexical processing. The primary finding suggests that in this normal study population, the visual cortices may be involved in the topographic spatial processing of TOR.

Mechanical ventilation for ischemic stroke and intracerebral hemorrhage: indications, timing, and outcome.

OBJECTIVE: To compare the incidence, indication, and timing of intubation and outcome in patients with cerebral infarction (ISCH) and intracerebral hemorrhage (HEM) requiring mechanical ventilation (MV). BACKGROUND: Poor outcomes have been reported for ISCH patients requiring MV. Because the target population, pathophysiology, and management of ISCH and HEM patients differ considerably, we compared the characteristics of patients with ISCH and HEM who required MV. METHODS: A retrospective review of ISCH and HEM stroke patients who underwent MV at a tertiary care academic center from 1994 to 1997 was performed to determine age, sex, type, and location of stroke (anterior or posterior circulation); brainstem dysfunction at intubation (pupillary, corneal, and oculocephalic reflexes); indication for intubation (neurologic deterioration, cardiopulmonary deterioration, or elective intubation for surgery); timing of intubation (on presentation or later); comorbidities; and outcome (hospital disposition). RESULTS: A total of 230 patients, 74 with ISCH and 156 with HEM (mean age, 61 +/- 16 years; male-to-female ratio, 1.15:1), underwent MV. Intubation rates were 6% for ISCH patients and 30% for HEM patients. Two-thirds of the patients required intubation on presentation (84% were intubated for neurologic deterioration) and 131 patients (57%) died (ISCH, 55%; HEM, 58%). Signs of brainstem dysfunction predicted a higher mortality for both groups. Additionally, early intubation and older age predicted mortality for HEM, and male gender predicted mortality in ISCH. Stroke location and comorbidities did not influence outcome. CONCLUSIONS: MV in acute stroke is associated with high mortality. Mortality and outcome were similar for ISCH and HEM; however, the factors predictive of outcome may differ and influence decisions about the use of MV in such patients.

Chronic Intrathecal Baclofen Administration for the Treatment of Severe Generalized Tetanus via a Synchromed Infusion Pump.

Centers for Disease Control (CDC) data indicate that the incidence of tetanus in the United States is highest among the elderly. Conventional therapies for the control of accompanying generalized muscle spasms include large doses of oral or intravenous GABA agonists as antispasticity agents. We describe a case of an elderly patient with severe symptoms of tetanus who developed a prolonged encephalopathy and ventilatory insufficiency with oral baclofen and benzodiazepine therapy. Intrathecal baclofen adequately controlled her severe extensor spasms, facilitated her ventilatory management, and did not compromise her mental status during her extended convalescence. Accordingly, we report the first placement of a permanent implanted infusion pump for this disease. This modality offers advantage for continuous long-term titration of medication for spasms or rigidity control without the systemic sedative effects of conventional therapy.

A case of CNS cryptococcosis presenting with slowly alternating skew deviation.

Alternating skew deviation is a rare malfunction of vertical ocular motility, which has previously been described with lesions of the brainstem and cerebellum, although the precise localization is unknown. We describe an HIV seronegative patient with CNS cryptococcosis, whose initial presentation included slowly alternating skew deviation.