Breast specimen orientation.

Lumpectomy specimens are commonly divided into six sides: superficial, deep, superior, inferior, medial, and lateral. Orienting stitches are placed on the specimen during surgery to allow reorientation by pathology. Despite those efforts, specimen disorientation may occur. The aim of this study was to assess the correlation in orientation between surgeons and pathologists. Lumpectomy specimens were routinely oriented. An additional Prolene suture was randomly placed by the surgeon on one side to be localized by pathology. The results were recorded and the disorientation rate calculated. Specimen size and presence of skin and/or muscle were also recorded. There were 122 lumpectomy specimens prospectively entered. Average specimen volume was 95.5 cm(3). Twenty-four specimens had segments of skin or muscle. The additional sutures were evenly divided between the six sides. The overall disorientation rate was 31.1% (95% confidence interval, 23.1-40.2).The side-specific disorientation rates were 43%, 40%, 35%, 29%, 28%, and 14% for the deep, superficial, lateral, medial, superior, and inferior surfaces, respectively (no statistical difference). Presence of skin or muscle on the specimen did not contribute to better orientation. Specimen volumes, however, were highly associated with orientation. Specimens of <20 cm(3) had a disorientation rate of 78%, while larger specimen had a disorientation rate of 20% (p < .001). Specimen orientation with stitches placed on two surfaces is associated with a high disorientation rate. Better orientation techniques are necessary to minimize the specimen disorientation.

Contemporary skin grafting in otologic surgery.

In the post-operative healing phase following external auditory canalplasty or open cavity mastoidectomy, problematic epithelialization of surfaces may ensue. Exposed surfaces or surface epithelialized with tissues other than skin may predispose to secondary infection and result in recurrent otorrhea. Split-thickness skin grafts can be useful in promoting healing of exposed canal and mastoid cavity surfaces. Our experience and methodology for harvesting and placing these grafts are reviewed. The indications, management, and complications of split-thickness otologic skin grafting will be presented.

Pilot scale production of pyrogen-free human hemoglobin for research.

A pilot scale production facility for the preparation of 20 to 30 liters of stroma-free hemoglobin is described. The system is capable of producing pyrogen-free solutions for research purposes. It is not certified for production of parenteral solutions for human use, but the plan could be implemented to meet standards for such materials. Products of the facility should be of adequate quality to address most of the toxicity and efficacy issues facing further development of hemoglobin-based red cell substitutes.

Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde.

Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5-phosphate (PLP) per heme and reduced with either NaCNBH3 or NaBH4. A separate sample was pyridoxylated and passed through a mixed-bed ion exchange column without reduction. All three preparations had a P50 of 29 +/- 2 torr and a cooperativity of n = 2.4 +/- 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4 degrees C and at room temperature. The polymerization invariably reduced the P50 to 18 +/- 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH3, all retained the PLP in differing amounts (2-3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the alpha- and beta-chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4 degrees C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH3 gave products with a lower P50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma-free hemoglobin, but a large number of molecular species were still formed.

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao.

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.

Preparation of human hemoglobin Ao for possible use as a blood substitute.

A procedure is presented for the preparation of a purified fraction of adult human hemoglobin (HbAo) from one unit of outdated blood. The entire process requires less than 16 h and gives a sterile, endotoxin-free solution of HbAo (approximately 30 g) in a yield of 50%. The solutions are isoionic with a conductivity of less than 15 mu mhos and less than 2 mmol total phosphorus per mol heme. The methemoglobin content is less than 1% and on storage at 4 degrees C rises less than 1% per month.

A convenient spectroscopic method for the estimation of hemoglobin concentrations in cell-free solutions.

Using a millimolar absorptivity of 7.12 +/- 0.09 at 523 nm, it is possible to estimate the total concentration of hemoglobin in solutions containing oxyhemoglobin, deoxyhemoglobin and methemoglobin in any combination. This estimate is independent of the pH in the range 6.0-10.0 and will provide concentrations comparable to that obtained by the conventional and more precise use of cyanomethemoglobin. This methodology should be of value for the determination of extracellular hemoglobin in vivo, as a means for determining the vascular half-life of stroma-free hemoglobin based blood substitutes.