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The lag period preceding the burst of oxygen consumption by phagocytes was measured with an oxygen electrode after the addition of unopsonized zymosan in CO-treated and diluted whole blood. The duration of the lag period correlated with the phagocytic index (r = 0.98, p < 0.001). The phagocytic index was defined as the mean number of zymosan particles per phagocyte. The lag period was termed opsonizing time and used for evaluation of plasma opsonizing activity. The opsonizing time was found to be inversely related to the plasma concentration used (< 15%) and was independent of phagocyte concentration. Magnesium ions were required throughout the opsonizing time, but calcium ions were essential only during the initial phase, indicating that the alternative complement pathway plays a major role in the observed opsonizing time. The mean opsonizing times in blood from normal adults and newborn infants were 5.6 +/- 0.7 min (n = 7) and 9.1 +/- 0.7 (n = 4), respectively. The opsonizing time, expressed as its reciprocal value, correlated significantly with phagocytic index, CH50 activity, and immunochemically assayed C3, but not C4, concentration in plasma.

The use of the interphase male Y-body (fluorescent Y chromosome segment) technique with cryostat sections of both fresh and frozen-stored skin biopsies is described. A female burn patient appeared to retain her donor homografts, thereby negating the need for autografts. Since a retained homograft of this sort challenged our understanding of immunologic barriers, we applied the Y-body technique to cutaneous biopsies obtained from the patient's burn area that had been homografted with skin from a male donor, as well as control biopsies from the patient's unburned skin and normal control male and female skin. Based on clinical and cytogenetic observations, it was concluded that the most reasonable explanation for this case was that the regenerating tissue at the graft site was that of the recipient and not that of the originally grafted male skin.

Computer vision techniques implemented on an IBM PC/AT have been applied to the study of microvascular permeability and interstitial diffusion in dorsal skin flap chamber preparations of hamsters. Experimental data was obtained for the leakage of fluorescent labelled dextran (70,000 daltons) after a precisely controlled mild degree of localized thermal trauma and compared with control data acquired prior to burn injury. Computer vision analysis techniques were applied to convert the fluorescent images into two-dimensional concentration maps. Interstitial diffusion coefficient values were computed from measured extravascular concentration profiles around a vessel of interest, assuming cylindrical or rectangular geometry, and optimally fitting a diffusion model to the data. An increase in the apparent diffusivity after mild thermal trauma was observed. Novel techniques were applied to solve hardware problems related to data acquisition and analysis, and a new library of software was developed to handle specific image processing requirements.

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbecco's minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.