Marcia K. Schmehl

Freeze-dried microarterial replacement allografts in rabbits

Microvascular techniques offer important alternatives for reconstructive head and neck surgery. To test the viability of freeze‐dried allografts, a pilot experimental study was performed using the rabbit model. Freeze‐dried preserved arterial allografts were implanted into femoral artery defects in eight subjects. After 6 weeks, all grafts were harvested and prepared for histologicol and electron microscopic analysis. Immediate patency was 100%. One subject was excluded on the third postoperative day. Of the seven remaining grafts, three (43%) were patent at 6 weeks. These results are comparable to previous data obtained using freeze‐dried arterial allografts in the some animal model. Although further investigation is required, this pilot study suggests possible future application of cryopreserved vascular micrografts.

Power analysis of statistical methods for comparing treatment differences from limiting dilution assays.

SummarySix different statistical methods for comparing limiting dilution assays were evaluated, using both real data and a power analysis of simulated data. Simulated data consisted of a series of 12 dilutions for two treatment groups with 24 cultures per dilution and 1,000 independent replications of each experiment. Data within each replication were generated by Monte Carlo simulation, based on a probability model of the experiment. Analyses of the simulated data revealed that the type I error rates for the six methods differed substantially, with only likelihood ratio and Taswells weighted mean methods approximating the nominal 5% significance level. Of the six methods, likelihood ratio and Taswells minimum Chi-square exhibited the best power (least probability of type II errors). Taswells weighted mean test yielded acceptable type I and type II error rates, whereas the regression method was judged unacceptable for scientific work.

Transplantation of cryopreserved canine venous allografts

Local vascular reconstructions frequently require the use of vein grafts to bridge arterial or venous defects. Most previous studies on the use of cryopreserved veins have used relatively large caliber vessels. There have been few studies on the effectiveness of cryopreserved micro- or small-venous allografts. Here, we tested two types of cryopreserved venous allografts: (1) 1.5- to 1.9-mm diameter microvenous grafts (MVG); and (2) 4- to 5-mm diameter small venous grafts (SVG). Cryopreserved MVG allografts were placed into saphenous arteries of six experimental dogs and SVG cryopreserved allografts were placed into femoral arteries of six experimental dogs for 3 to 6 weeks. Two fresh MVG autografts were also transplanted into experimental dogs as controls and autografts were transferred to the contralateral side in SVG dogs as controls. None of the six cryopreserved MVG grafts retained patency but three/six cryopreserved SVG allografts were patent at harvest. Histological examination of grfts revealed control autografts were undergoing arterialization with an intact intima. Experimental cryopreserved allografts showed extensive medial fibrosis, significant lymphocytic infiltrates, and sporadic areas of intact intima for both patent and nonpatent grafts.

Comparison of statistical methods for the analysis of limiting dilution assays.

SummaryThis study reports the results of a critical comparison of five statistical methods for estimating the density of viable cells in a limiting dilution assay (LDA). Artificial data were generated using Monte Carlo simulation. The performance of each statistical method was examined with respect to the accuracy of its estimator and, most importantly, the accuracy of its associated estimated standard error (SE). The regression method was found to perform at a level that is unacceptable for scientific research, due primarily to gross underestimation of the SE. The maximum likelihood method exhibited the best overall performance. A corrected version of Taswells weighted-mean method, which provides the best performance among all noniterative methods examined, is also presented.

Confidence Intervals for the Relative Frequency of Responding Cells in Limiting Dilution Assays

SUMMARY Confidence intervals for the relative frequency of responding cells in limiting dilution assays (LDAs) have not been examined closely. Generally, confidence intervals are calculated by using the normal approximation, sometimes preceded by a log transform. We evaluate and compare with analytical approaches: the normal approximation, the log transform, and two binomial methods-the quadratic approximation and a modification of the Clopper-Pearson exact method. We evaluate these confidence interval methods for use with the maximum likelihood and jackknife estimators of the relative frequency. Our results show that confidence interval construction with maximum likelihood point estimates is more accurate than construction with jackknife estimates. When using maximum likelihood estimates, the normal approximation produces acceptable two-sided confidence intervals, with the smallest length, at a = .05. At a = .01, the normal approximation is anticonservative. In all cases, the normal approximation is unable to produce adequate one-sided confidence intervals. The log transform and both binomial confidence interval methods are shown to be generally superior to

Endothelial and fibroblast viability assays for tissue allografts

To perfect methods for the freezing, storage and subsequent transplantation of cells and tissues, it is important to set the criteria for success at the outset. Therefore one or more criteria should be defined which accurately maintain the ability of the system to carry on its physiological function. For example, a frozen-thawed vein should be capable of performing as a conduit after implantation. The vein should not be prone to stenosis, aneurysms, or leakage around the suture lines, and should ideally be non-thrombogenic. Since thrombosis and vascular tone is dependent upon the presence of an intact endothelial lining, a viable cryopreserved allograft should have an intact endothelial lining. For heart valves, the presence of a high percentage of the fibroblasts which are capable of resynthesizing the collagenous matrix of the valve, as well as maintaining the mechanical integrity, is the primary consideration. The viability of any tissue after cryopreservation is dependent in part upon handling during procurement and prefreezing storage. Any exposure to non-physiological conditions, such as ischemia, hypoxia, or anoxia causes direct toxicity to most cell types or sensitizes the cells to the subsequent stresses of freezing and thawing. Careful selection of the cryobiological variables can minimize but not eliminate the loss in viability. Major considerations include the type of cryoprotective agent used, the concentration of that agent, the temperature of exposure, cooling rate, warming rate, osmotic effects, media effects, and dilution scheme.

Comparative characteristics of estimators of the mean responding cell density in limiting dilution assays

The statistical problems associated with estimating the mean responding cell density in the limiting dilution assay (LDA) have largely been ignored. We evaluate techniques for analyzing LDA data from multiple biological samples, assumed to follow either a normal or gamma distribution. Simulated data is used to evaluate the performance of an unweighted mean, a log transform, and a weighted mean procedure described by Taswell (1987). In general, an unweighted mean with jackknife estimates will produce satisfactory results. In some cases, a log transform is more appropriate. Taswells weighted mean algorithm is unable to estimate an accurate variance. We also show that methods which pool samples, or LDA data, are invalid. In addition, we show that optimization of the variance in multiple sample LDAs is dependent on the estimator, the between-organism variance, the replicate well size, and the numberof biological samples. However, this optimization is generally achieved by maximizing biological samples at th...

Evaluation and validation of statistical methods for viability assays: Monte Carlo simulation and power analysis of limiting dilution assay data

Scientists frequently must choose between several different statistical methods for analyzing data. Monte Carlo simulations and power analyses can be used to select the most appropriate statistical method for analyzing viability assay data and to choose the statistical method that best differentiates between small differences in cryopreservation treatments. In addition, the probability of Type I and Type II statistical errors and the relationship between Type I and Type II errors can also be determined by these techniques. Monte Carlo results for six methods of analyzing limiting dilution assay data revealed discrepancies between theoretical and true significance levels for test methods. The methods tested were regression, weighted means, unweighted means, maximum likelihood, minimum X2 and 2 × 2 X2. The maximum likelihood method was found to be superior to other methods because it (i) tested closest to the theoretical significance level, and (ii) had the least bias, the lowest sampling variation, and the most power for determining differences between treatments.

Evaluating the mean frequency of responding cells in limiting dilution assays

SummaryThe validity of limiting dilution assays can be compromised or negated by the use of statistical methodology which does not consider all issues surrounding the biological process. This study critically evaluates statistical methods for estimating the mean frequency of responding cells in multiple sample limiting dilution assays. We show that methods that pool limiting dilution assay data, or samples, are unable to estimate the variance appropriately. In addition, we use Monte Carlo simulations to evaluate an unweighted mean of the maximum likelihood estimator, an unweighted mean based on the jackknife estimator, and a log transform of the maximum likelihood estimator. For small culture replicate size, the log transform outperforms both unweighted mean procedures. For moderate culture replicate size, the unweighted mean based on the jackknife produces the most acceptable results. This study also addresses the important issue of experimental design in multiple sample limiting dilution assays. In particular, we demonstrate that optimization of multiple sample limiting dilution assays is achieved by increasing the number of biological samples at the expense of repeat cultures.