Reduction of intra- and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training. DK34 Task Force of the European Working Group of Clinical Cell Analysis (EWGCCA)

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single-platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non-standardized methods. Two bead-counting systems, Flow-Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow-Count, the intralaboratory coefficient of variation (CV) was 相似文献   

The allatotropic activity of the larval brain of Galleria mellonella cultured in vitro

The brains of last instar larvae of Galleria mellonella can be successfully cultured in vitro. The high allatotropic activity of 0–8 hr brains, as measured by their ability to elicit supernumerary larval molts upon implantation into 0–8 hr last instar larval hosts, remains constant through 4 days in vitro. The allatotropic activity of 120 hr brains increases during in vitro culture. The number of supernumerary larvae resulting from the implantations of 120 hr brains rises from 20 to 30% of the hosts implanted with fresh brains, to 90% of the hosts supplied with brains maintained in vitro for 24 hr prior to implantation. There is a progressive loss of paraldehyde-fuchsin-stainable neurosecretory material over 72 hr of in vitro culture from the three groups of neurosecretory cells visible in whole mounts of the brains. Nevertheless, the ultrastructural picture of the medial neurosecretory cells, which are the probable source of the allatotropic factor, indicates synthetic activity after 72 hr of culture. In 0–8 hr last instar brains which are cultured for 48 hr as complexes with attached corpora cardiaca and corpora allata, stainable neurosecretory material accumulates in all three groups of cerebral neurosecretorycells. The brains from the cultured complexes have low allatotropic activity. It is concluded on the basis of the bioassay and ultrastructural observations that under our conditions of in vitro culture, the neurosecretory cells of the last instar larval brain not only survive but also maintain synthetic activity through at least 72 hr in vitro. The relationship between stainable neurosecretory material and neurosecretory activity in this system is discussed, as are possible feedback interactions involving the corpora cardiaca-corpora allata and the brain.     

Placental trophoblast-derived factors diminish endothelial barrier function

Although increased vascular permeability is an important event in the pathogenesis of preeclampsia, the origin of the circulating factor(s) that elicits this endothelial barrier dysfunction is not known. In this study, we use coculture of endothelial cells and placental trophoblast cells to determine whether placental trophoblasts are a potential source of the factor(s) that mediate the increased vascular permeability of preeclampsia. Human umbilical vein endothelial cells grown in Transwell inserts or on coverslips were cocultured with trophoblast cells isolated from normal and preeclamptic placentas or placenta conditioned media. Endothelial cell barrier function was determined by: 1). measurements of electrical resistance and leakage of horseradish peroxidase, and 2). immunofluorescent staining of vascular endothelial-cadherin, pan-cadherin, and occludin. Uterine myometrium endothelial cells were also studied for comparison. We observed the following: 1). electrical resistance was significantly (P < 0.01) decreased (compared with control endothelial cells) in endothelial cell monolayers cocultured with normal trophoblast cells and further reduced in endothelial cells cocultured with preeclamptic trophoblast cells; 2). an increased horseradish peroxidase leakage that was correlated with the decreased electrical resistance in cocultured cells; and 3). disorganized tight junction proteins and an altered distribution of vascular endothelial-cadherin and occludin in monolayers of endothelial cells cocultured with preeclamptic trophoblast cells. Similar responses were noted in uterine myometrium endothelial cells. We conclude that: 1). placental trophoblast cells produce factors that diminish the barrier function of endothelial cells; 2). endothelial tight junctions are more susceptible to factors released from preeclamptic trophoblast cells than from normal trophoblast cells; and 3). these results implicate trophoblast-derived factors in the increased vascular permeability associated with preeclampsia.     

In vitro activation of insect prothoracic glands by the prothoracicotropic hormone

An in vitro assay for the prothoracicotropic hormone has been developed that utilizes an ecdysone radioimmunoassay to quantify the increase in the rate of ecdysone synthesis elicited by the neurohormonal activation of the prothoracic glands. The rapidity, reproducibility, and accuracy of the assay were maximized by using one member of a gland pair as the control and the other as the test gland. This was possible because the basal rates of ecdysone synthesis by the members of a gland pair were equivalent. Activation was demonstrated to be dose dependent and specific, with prothoracicotropic hormone activity present only in homogenates of brain. The in vitro activation of the prothoracic glands was verified with the Manduca bioassay for the prothoracicotropic hormone in which the morphological responses to the hormone were correlated with increased in vivo ecdysone titers. These results provide unequivocal evidence that the activation of the prothoracic glands by the prothoracicotropic hormone is direct and suggest that activation represents an increase in a basal rate of ecdysone synthesis.     

One-year Patient-reported Outcomes After Arthroscopic Rotator Cuff Repair Do Not Correlate With Mild to Moderate Psychological Distress

Background

Patients with shoulder and rotator cuff pathology who exhibit greater levels of psychological distress report inferior preoperative self-assessments of pain and function. In several other areas of orthopaedics, higher levels of distress correlate with a higher likelihood of persistent pain and disability after recovery from surgery. To our knowledge, the relationship between psychological distress and outcomes after arthroscopic rotator cuff repair has not been similarly investigated.

Questions/purposes

(1) Are higher levels of preoperative psychological distress associated with differences in outcome scores (visual analog scale [VAS] for pain, Simple Shoulder Test, and American Shoulder and Elbow Surgeons score) 1 year after arthroscopic rotator cuff repair? (2) Are higher levels of preoperative psychological distress associated with less improvement in outcome scores (VAS for pain, Simple Shoulder Test, and American Shoulder and Elbow Surgeons score) 1 year after arthroscopic rotator cuff repair? (3) Does the prevalence of psychological distress in a population with full-thickness rotator cuff tears change when assessed preoperatively and 1 year after arthroscopic rotator cuff repair?

Methods

Eighty-five patients with full-thickness rotator cuff tears were prospectively enrolled; 70 patients (82%) were assessed at 1-year followup. During the study period, the three participating surgeons performed 269 rotator cuff repairs; in large part, the low overall rate of enrollment was related to two surgeons enrolling only two patients total in the initial 14 months of the study. Psychological distress was quantified using the Distress Risk Assessment Method questionnaire, and patients completed self-assessments including the VAS for pain, the Simple Shoulder Test, and the American Shoulder and Elbow Surgeons score preoperatively and 1 year after arthroscopic rotator cuff repair. Fifty of 85 patients (59%) had normal levels of distress, 26 of 85 (31%) had moderate levels of distress, and nine of 85 (11%) had severe levels of distress. Statistical models were used to assess the effect of psychological distress on patient self-assessment of shoulder pain and function at 1 year after surgery.

Results

With the numbers available, distressed patients were not different from nondistressed patients in terms of postoperative VAS for pain (1.9 [95% confidence interval {CI}, 1.0–2.8] versus 1.0 [95% CI, 0.5–1.4], p = 0.10), Simple Shoulder Test (9 [95% CI, 8.1–10.4] versus 11 [95% CI, 10.0–11.0], p = 0.06), or American Shoulder and Elbow Surgeons scores (80 [95% CI, 72–88] versus 88 [95% CI, 84–92], p = 0.08) 1 year after arthroscopic rotator cuff repair. With the numbers available, distressed patients also were not different from nondistressed patients in terms of the amount of improvement in scores between preoperative assessment and 1-year followup on the VAS for pain (3 [95% CI, 2.2–4.1] versus 2 [95% CI, 1.4–2.9], p = 0.10), Simple Shoulder Test (5.2 [95% CI, 3.7–6.6] versus 5.0 [95% CI, 4.2–5.8], p = 0.86), or American Shoulder and Elbow Surgeons scale (38 [95% CI, 29–47] versus 30 [95% CI, 25–36], p = 0.16). The prevalence of psychological distress in our patient population was lower at 1 year after surgery 14 of 70 (20%) versus 35 of 85 (41%) preoperatively (odds ratio, 0.36; 95% CI, 0.17–0.74; p = 0.005).

Conclusions

Mild to moderate levels of distress did not diminish patient-reported outcomes to a clinically important degree in this small series of patients with rotator cuff tears. This contrasts with reports from other areas of orthopaedic surgery and may be related to a more self-limited course of symptoms in patients with rotator cuff disease or possibly to a beneficial effect of rotator cuff repair on sleep quality or other unrecognized determinants of psychosocial status.

Level of Evidence

Level I, prognostic study.     

Hemostatic plug formation in normal and von Willebrand pigs: the effect of the administration of cryoprecipitate and a monoclonal antibody to Willebrand factor

Hemostatic plug (HP) formation was investigated in the ear bleeding time incision in normal and von Willebrand pigs. HP volume was calculated by integrating the areas of serial sections. In normal pigs (n = 11), platelets immediately formed a layer on the surface of the cut channel. Platelet aggregates formed at the ends of transected vessels and gradually enlarged. Finally, all transected vessels were occluded by HP and bleeding stopped. In contrast, large HPs were formed in the incision in von Willebrand's disease (vWD) pigs (n = 4); these HPs did not cover the ends of the transected vessels, which continued to bleed, allowing the formation of large hemostatically ineffective platelet aggregates in the incision. Canals traversed these HPs, and bleeding from the open vessels may have continued through them. After infusion of cryoprecipitate into a vWD pig, the bleeding time shortened, and the morphological findings of the HPs were similar to those of normal pigs. In normal pigs (n = 3) infused with an anti- Willebrand factor monoclonal antibody, which prolonged the bleeding time, a large HP formed in the incision, similar to that observed in the vWD pig. The volume of the normal and vWD HPs increased with time. These in vivo findings suggest that Willebrand factor is involved in the localization of the HP to the damaged vessel and may also play a role in platelet-platelet interaction. A computerized morphometric technique was used for measuring the volume of the hemostatic plugs and the distance of sequential points on the perimeter of the HP from the center of selected bleeding vessels.