The impact on tissue utilization of screening donor corneas by specular microscopy at the University of Texas Southwestern Medical Center.

To determine how frequently specular microscopy results affect the outcome of eye-bank judgments on the transplantability of donor corneas, 1,011 consecutive donor records from a 3-year period at the Transplant Services Center of the University of Texas Southwestern Medical Center at Dallas were analyzed. Specular microscopy cell counts from each decade of donor age were determined, and it was found that there were no cell counts < 2,000 mm2 for any donor age < 40. Above age 40, the percentage of cell counts < 2,000 per mm2 rose from 3.9% for donors in their forties of 6% for donors in their seventies. For donors between 40 and 69 years, specular microscopy was used to rule out unacceptable tissue in an additional 3.3% of a prescreened pool of corneas evaluated by current Eye Bank Association of America standards. While corneas from donors over age 69 were initially presumed to be unacceptable for transplant at this eye bank, routine specular microscopic examination helped to clear for transplant of 31 corneas from donors of this age group.

Guidelines for preventing "dilution false negatives" in in vitro laboratory testing of the donor population.

In this article we develop a calculation or formula for use in determining the potential dilution effect of fluids administered during patient treatment on serologic testing parameters. The formula uses basic principles of (a) fluid distribution over time from administration; (b) ratios of plasma and extravascular fluid volumes to body weight; and (c) common practices of fluid resuscitation. A dilution threshold of 50% was set using data from enzyme-linked immunosorbent assay human immunodeficiency virus antibody determinations performed on in vitro diluted seropositive serum samples. These data respond to issues raised by guidelines from the Centers for Disease Control and the U.S. Food and Drug Administration to achieve recipient safety without unnecessarily restricting the potential donor pool.

Composite skin graft: frozen dermal allografts support the engraftment and expansion of autologous epidermis.

Rapid closure of burn wounds significantly reduces the complications associated with thermal injury. Successful wound coverage, however, is often limited by the lack of suitable autografts. To circumvent this limitation a composite graft was developed which combines the utility and availability of allogeneic skin with the permanence of an autograft. Composite grafts were first employed in a rat wound model and subsequently to treat six patients with thermal injuries. In experiments with rats, full-thickness excised (1") wounds were prepared on thoracic walls, covered with previously frozen allograft skin, dressed, and secured. Five days later, the dead epidermis was removed and trypsin-disaggregated syngeneic epidermal cells applied to the exposed dermal surface. Successful engraftment with complete epidermal coverage could be observed within 7 to 10 days. In eight patients, split-thickness skin bank allografts were placed on full-thickness burn wounds. Four days later the dead epidermis was removed and vacuum blister-prepared sheets of autologous epidermis grafted to the exposed dermal surface. In all eight patients successful engraftment ensued. Increased pigmentation at the site of each original epidermal graft confirmed the stability of underlying allograft dermis. Epidermal expansion ranged from 1:20 to 1:100. All patients were followed from 10 to 12 months with no demonstrated graft loss or significant wound contracture. Composite skin grafts which combine allogeneic dermis and an expanded autologous epidermis can effect rapid wound closure and will remain stable without evidence of rejection or graft breakdown for at least 12 months.

Identification of nonviable muscle in electric burns with nitroblue tetrazolium.

Both experimental and clinical evaluation of nitroblue tetrazolium (NBT) as a method of identifying ischemic and necrotic muscle is described. Colorless NBT was reduced to a blue formazin by viable muscle but remained colorless in necrotic muscle. Muscle samples were rated for viability on a scale of 0-100% based on the relative amount of muscle sample that reduced NBT. There was good correlation between gross appearance, distribution of staining, and histologic findings in all nonviable experimental and clinical muscle specimens. All tissue that reacted with NBT proved to be viable histologically. Metabolically active or viable muscle fibers are rich in respiratory enzymes (dehydrogenases). The loss of dehydrogenase activity from ischemic or necrotic muscle can be detected by NBT reduction capacity. NBT technique identified nonviable tissue, clearly showed the even distribution of muscle damage characteristic of electric burns, was useful in defining ischemic muscle prior to obvious necrosis, and was a rapid, simple, and reliable semiquantitative test that can be used intraoperatively.

Structure-volume relationships: singular volume effects produced by cupric ion-globular protein interaction.

The nature of the volume isotherms produced by the coordination of Cu(II) with ovalbumin and bovine serum albumin differs substantially from the adsorption isotherms produced by these systems. Whereas there was increased binding of Cu(II) associated with a pH increase from pH 5.3 to pH 7.4, the volume isotherms for these systems did not exhibit this type of pH dependence. The volume changes were determined at 30.0 +/- 0.001 degrees C with microdilatometers which could be read to 0.01 muL. The binding isotherms for ovalbumin at pH 5.3 and 7.4 and for bovine serum albumin at pH 5.3 was resolved by a Scatchard plot to yield the appropriate thermodynamic parameters. An algorithm was derived to calculate the distribution of the individual PMi complexes, i.e., PMi-1 + M in equilibrium (Ki) PMi where i equals 1, 2, 3, ..., n moles of cation, M, bound per mole of protein, P, for the above systems. The volume isotherms were then resolved in terms of the constituent delta Vi terms, i.e., the volume change produced by the formation of the individual PMi complexes. These values were verified by an independent graphical differentiation procedure. The coordination of Cu(II) to BSA at pH 7.4 produced a cooperative adsorption isotherm which was not amenable to a Scatchard analysis. The resultant anomalous volume isotherm was resolved into a component related to Cu(II)-site interaction and a negative volume effect attributable to a conformational change induced by complex formation. This structural transition which occurs at physiological pH may constitute a control mechanism for regulating the serum level of Cu(II) and possibly other divalent ions.

A critical evaluation of povidone-iodine absorption in thermally injured patients.

Povidone-iodine ointment is a widely used topical antimicrobial agent in thermally injured patients. In 17 patients with burns ranging from 4 to 85% TBSA this agent was applied to both partial- and full-thickness burns. Peak serum iodine levels in patients treated within 24 hours of injury ranged from 595 to 4,900 micrograms per dL. The amount of iodine absorbed was directly related to the size of the burn. Serum iodine levels continued to rise until the drug was discontinued and remained elevated for as long as 7 days after discontinuance. Iodine excretion was directly related to renal function. The highest serum and lowest urinary iodine levels were present in patients who developed renal failure. Thyroid function was not affected by drug usage. A proven cause-and-effect relationship between the elevated serum iodine attributed to the absorption of povidone-iodine and metabolic acidosis, hypernatremia, and hyperosmolarity was not established. Close monitoring of the patient's fluid and electrolyte balance is imperative during drug usage. The drug is contraindicated in any patient with impaired renal function. The high serum levels of this inorganic ion imply potential toxicity, but clinical evidence of cell or organ toxicity is as yet undetermined.

The role of NADH-NADPH oxidase activity in the leukocyte function of burned patients.

Irreversible sepsis, in spite of advancements in topical therapy and antimicrobial agents, remains the leading cause of death in major thermal injury. A defect in intracellular bactericidal capacity in leukocytes from severely burned patients appears to correspond with increases in bacterial wound colonization and ultimate sepsis. This leukocyte defect has been demonstrated by abnormally low nitroblue tetrazolium reduction (NBT) and oxygen consumption of white cells in patients with major thermal injury. The subcellular mechanisms responsible for decreased bactericidal capacity were therefore investigated. Nicotinamide-adenine dinucleotide (NADH) and nicotinamide-adenine phosphodinucleotide (NADPH) oxidase activity was measured in patients with major burns, controls (normals), and in patients with nonburn stress or infection. NADH and NADPH oxidase levels in leukocytes from burn patients were not significantly different from those of normal nonchallenged controls but were significantly lower than the leukocyte values found in the patients with nonburn infections or stress. This NADH and NADPH defect in the subcellular leukocyte fraction suggests that it may be a significant factor in the reduced bactericidal function of the intact leukocyte in thermally injured patients.

Usefulness of blood cultures in confirming septic complications in burn patients: evaluation of a new culture method.

A prospective evaluation of several techniques used to monitor thermally injured patients for septic complications was initiated. Thirty-eight extensively burned patients, at high risk for septic complications, were selected for study. Of these, 92% developed significant burn wound infection. Careful daily clinical evaluation and serial quantitative burn wound biopsy cultures provided the most effective means of establishing an early diagnosis of would sepsis. Serial blood cultures performed by the two different techniques were of very limited value in the recognition were not recovered until 5-10 days following documentation of heavy bacterial colonization of the burn wound.