Validation and reproducibility of computerised cell-viability analysis of tissue slices

Background

The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results.

Methods

To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes.

Results

Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77).

Conclusions

This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

     

MASTOS: Mammography Simulation Tool for design Optimization Studies

Mammography is a high quality imaging technique for the detection of breast lesions, which requires dedicated equipment and optimum operation. The design parameters of a mammography unit have to be decided and evaluated before the construction of such a high cost of apparatus. The optimum operational parameters also must be defined well before the real breast examination. MASTOS is a software package, based on Monte Carlo methods, that is designed to be used as a simulation tool in mammography. The input consists of the parameters that have to be specified when using a mammography unit, and also the parameters specifying the shape and composition of the breast phantom. In addition, the input may specify parameters needed in the design of a new mammographic apparatus. The main output of the simulation is a mammographic image and calculations of various factors that describe the image quality. The Monte Carlo simulation code is PC-based and is driven by an outer shell of a graphical user interface. The entire software package is a simulation tool for mammography and can be applied in basic research and/or in training in the fields of medical physics and biomedical engineering as well as in the performance evaluation of new designs of mammography units and in the determination of optimum standards for the operational parameters of a mammography unit.     

Anticardiolipin antibodies and occlusive vascular disease of the eye: Prospective study

There is a recognized association between the presence of anticardiolipin antibodies and vascular occlusive disease. The purpose of our study is to detect the presence of high titers of anticardiolipin antibodies (ACA) in the serum and to correlate the titers with the severity of the vascular disease in patients with occlusive ocular vascular disease. 82 patients were included in a prospective study; 25 patients with anterior ischaemic optic neuropathy, 36 with retinal vein occlusion and 21 with retinal artery occlusion. ACA (IgG and IgM isotypes) were measured by ELISA in the sera of all patients. The group of the patients (group A) was compared to an age-matched control group of 79 healthy individuals (group B). IgG isotype (but not IgM) titers of ACA were found significantly higher in group A (P < 0; 0.001). In patients with titers of ACA (IgG isotype) > 100 units we noted a higher incidence of a more severe disease (recurrency, involvement of both eyes or extraocular manifestations) especially among those with anterior ischaemic optic neuropathy and secondarily in those with retinal artery occlusion. Our results suggest that the association between high titers of ACA and occlusive vascular disease of the eye concerns only the IgG isotype. In addition, the detection of high titers of IgG/AGA in patients could be a useful marker for disease severity and prognosis and this observation seems to be more explicit in cases with arterial occlusive disease than in cases with venous occlusive disease. This revised version was published online in July 2006 with corrections to the Cover Date.     

Iron, ascorbate and copper status of Sowetan Blacks with calcific chronic pancreatitis

Vitamin C can be used to overcome oxidative stress and ease pain in chronic pancreatitis. But its use is deprecated in conditions of tissue iron overload, because its bioactive form, ascorbate, can accelerate free-radical reactions that are driven by transition metals. We measured iron, ascorbate and copper in Sowetan Blacks (RSA) with chronic pancreatitis, obtaining serum/plasma from 14 consecutive patients and 15 controls. Compared with data from corresponding groups in Manchester, African samples had less ascorbate (p < 0.0001), but more caeruloplasmin (p < 0.0001). African and British controls had comparable iron and iron-binding capacity. Plasma from African patients had less ascorbate than that from African controls (p < 0.005) and in six samples, ferritin exceeded 300 micrograms/l (677 pmol/l). Low- molecular-mass iron or copper, capable of participating in free radical reactions, was not detected. British patients, had similar caeruloplasmin levels to African patients but higher ascorbate levels. There is no evidence of iron overload in our African samples. Outwardly healthy controls from Soweto have elevated levels of caeruloplasmin, possibly to compensate for dietary deficiency of ascorbate. Persistent oxidative stress is a unifying feature of chronic pancreatitis, but its degree is higher in African than British patients. Supplements of vitamin C should be safe in Blacks of southern Africa.      

Physiological interpretation of electrical impedance epigastrography measurements

Measurement of the electrical impedance of the gastric region is carried out with the epigastrograph. This generates and applies alternating current around the abdominal area and measures the potential difference in order to determine the impedance externally, via electrodes. The change of epigastric impedance for a subject, given a meal after fasting, depends on the conductivity of the meal compared to the stomach and surrounding tissues. Typically a conductive meal has conductivity >7 mS cm(-1), non-conductive <2 mS cm(-1) and neutral about 4.5 mS cm(-1). Half-emptying times (T50s) from gastric emptying studies in volunteers using three test meals of 450 ml volume were obtained and found to be shorter than expected from the literature. The meals were a 10% glucose solution and two milk shakes of energy 1,300 kJ and 2,850 kJ, respectively. These electrical impedance epigastrography (EIE) measurements were carried out with scintigraphy. The T50 values of the latter were significantly longer. The direct comparison of the normalized experimental data obtained by both methods led to the concept that EIE measurements are mainly influenced by gastric secretion. Thus the EIE trace of a 'neutral' meal suggests the hypothesis that the volume of the meal is not the significant factor but is influenced by gastric acid secretions. Physiology of the gastric mucosa during the digestion of a meal and intragastric pH values also suggests this. Gastric function studies using EIE measurements may therefore reflect gastric ionic concentration rather than the volume of the contents of the stomach. In turn this could lead to the development of a non-invasive method for the continuous recording of gastric acid secretions.     

Hypergammaglobulinemia can be associated with a positive direct antiglobulin test, a nonreactive eluate, and no evidence of hemolysis

To determine the cause of a positive direct antiglobulin test (DAT), blood banks routinely perform serologic tests on eluates prepared from DAT-positive red cells. Negative eluates traditionally have been suspected to be associated with drug reactions. This report confirms that the most frequent cause of a positive DAT and a nonreactive eluate is hypergammaglobulinemia. The results of 74 patient samples with positive DATs were analyzed retrospectively. Eluates prepared from the red cells of 54 patients (72.9%) reacted; eluates from 20 patients (27.1%) did not react. This latter group had identical serologic and clinical findings, suggesting that they made up a homogeneous group. In particular, the patients had a positive DAT, a negative indirect antiglobulin test, and a negative eluate; an increased serum concentration of IgG; and no evidence of hemolysis. In a subsequent study, DATs were performed prospectively on red cells from 44 consecutive patients with elevated serum IgG levels. The serum IgG concentration was highest in the three patients whose red cells had a positive DAT. The DAT also became positive in two patients treated with high-dose intravenous gammaglobulin (IV IgG). These studies indicate that a negative eluate from red cells with a positive DAT, a common serologic finding, is often caused by hypergammaglobulinemia. The authors postulate that IgG binds nonspecifically to the red cells because of the hypergammaglobulinemia.     

QUADAS评价:一种用于诊断性研究的质量评价工具(修订版)

背景 QUADAS是一种新近发展起来的诊断性研究的质量评价工具。虽然已有系统评价采用了QUADAS,但尚未得以正式确证。本研究的目的是评价QUADAS的有效性和实用性。方法3位评价者采用QUADAS独立评价30项研究的质量。比较每位评价者的评分与最终结论之间的意见一致度。这主要是通过比较所有QUADAS条目的总分和每个单项的得分来实现的。20位曾在其系统评价中使用过QUADAS的评价者就其使用经验完成一份简短的问卷。结果就所有条目而言,每位评价者的评分与最终结论之间的意见一致度分别达到了91%、90%和85%。就单项QUADAS条目而言,一致度在50%至100%之间,中位值是90%。与难以解释的试验结果和退出病例相关的条目,评分结果差异最大。有关QUADAS内容的反馈意见普遍较好,仅少数评价者提出了有关QUADAS涵盖面、使用便利性、评分说明的清晰度及有效性方面的问题。结论QUADAS内容本身无需作大的修改。评价过程的主要困难出现在难以解释的试验结果和退出病例这2个条目的评分上。对这些条目的评分指南提出了修改意见。评价者必需根据其系统评价制定相应的评分指南,并确保所有评价者都清楚如何评分。评价者还应考虑是否所有的QUADAS条目都与其系统评价相关,以及其他质量条目是否应作为其系统评价的评价部分。     

Winning the battle but losing the war: methicillin-resistant Staphylococcus aureus (MRSA) infection at a teaching hospital

A methicillin-resistant Staphylococcus aureus (MRSA) control policy, aimed at eradication, was established at a 1000-bed hospital in 1985, applied consistently for 10.5 years, and then relaxed. Its components included screening of high-risk patients, transfer of carriers to exhaust-ventilated isolation rooms, closure of wards to new admissions when local transmission was detected, MRSA screening during outbreaks, and prospective collection of clinical and epidemiological information. During the eradication policy period, every 6 months, a mean of 5.1 patients (range 1-12) already carrying MRSA were admitted, and a mean of 3.6 (range 0-16) acquired carriage in the hospital. The largest outbreak comprised 11 patients despite epidemic MRSA strain EMRSA-16 being introduced six times, and MRSA did not become endemic. MRSA- positive admissions increased progressively from 1993; nursing staff workload increased, areas available for alternative patient accommodation were reduced, the resulting ward closures interfered with clinical services, and hence the control policy was relaxed in mid- 1995. Isolation facilities were overwhelmed with 622 new patient- isolates in the next 18 months, and there were 67 clinical infections in 1996. The proportion of blood cultures positive for MRSA rose nearly sevenfold by 1996 and 27-fold by 1997. Thus, repeated eradication of MRSA, even epidemic strains, by use of a stringent policy, is possible given sufficient resources, whereas flexible national guidelines designed to control, but not eradicate, epidemic staphylococci, are currently unlikely to be successful. The costs of eradication policies need to be weighed against those of endemicity.      

Sentinel lymph node biopsy in melanoma: Which hot nodes should be harvested and is blue dye really necessary?

Objectives

The ‘10% rule’ has become widely accepted by surgeons performing sentinel lymph node biopsy (SLNB) for melanoma. The purpose of this study was to compare the ‘10% rule’ with alternative node harvesting criteria. In particular, we were interested to see whether the use of blue dye had any impact on the sensitivity of the test and whether it is necessary to remove all hot nodes.