Lowell L. Tilzer

Use of silica gel polymer for DNA extraction with organic solvents

Phenol and chloroform are the standard solvents used for DNA extraction. These solvents aid in the removal of protein and lipid from crude or partially purified cell extracts. Although the procedure is well established, the solvents are noxious, caustic, and unpleasant. We describe in this paper the use of a special blood collection tube to isolate the offensive organic solvents. With the use of silica gel polymer containing tubes, phenol, phenol:chloroform, or chloroform can be separated from the DNA containing aqueous phase in a rapid and safe manner. The method permits higher yields of DNA since the DNA is poured from the tube rather than aspirated with pipet.

Identification of donor melanoma in a renal transplant recipient.

A patient with chronic renal failure received a closely matched cadaveric kidney. Approximately 3 months after transplantation, the patient developed a metastatic malignant melanoma. A large retroperitoneal mass consisting of large pleomorphic polygonal neoplastic cells was found close to the donated kidney. This tumor was diagnosed as a malignant melanoma. DNA analysis of this tumor, the donated kidney, and the recipient indicated that the melanoma originated from the donor. Although this is not the first report of a donated melanoma, it is the first report of definitive DNA analysis of the origin of the malignant cells.

Effect of high-dose of methylprednisolone on tourniquet ischaemia

High doses of corticosteroids have been found to have beneficial effects in various shock states. It has been well recognized that ischaemia is one of the important features in shock states. This prompted us to investigate the effect of high-dose methylprednisolone on tourniquet-induced ischaemia using mongrel dogs. After inflation of tourniquets to 600 mmHg on each thigh of the hind legs, one leg received an intravenous infusion of methylprednisolone, 3 mg · kg-1 dissolved in 20 ml of autologous blood. The other leg received the same amount of blood only, as a control. During two hours of tourniquet time and until 30 min after tourniquet deflation, venous blood was sampled five times from both hind legs for measurements of blood gas tensions (PvO2, PvCO2) and pH, lactic acid, creatinine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). During tourniquet ischaemia, PvO2 and pH dropped and PvCO2, lactic acid, CK, AST and LDH rose steadily and significantly in both groups of legs, indicating respiratory and metabolic acidosis, and muscle cell damage. However, those changes were significantly smaller in the methylprednisolonetreated legs. The beneficial effect of methylprednisolone could be attributed to its vasodilatory effect, cellular membrane stabilization and direct metabolic effect on skeletal muscle cells. Although the tourniquet-induced ischaemia in our study is slightly different from the clinical paradigm, the results suggest that high-dose methylprednisolone may provide a beneficial effect during tourniquet ischaemia.RésuméDes doses élevées de corticostéroïdes ont été trouvées bénéfiques dans différents états de choc. Il est bien reconnu que ľischémie est un signe important des états de choc. Ceci nous a conduit à investiguer ľeffet de doses élevées de méthylprednisolone sur ľischémie induite par le tourniquet chez les chiens bâtards. Après inflation du tourniquet à 600 mmHg sur chaque cuisse des pattes arrière, une patte a reçu la perfusion intraveineuse de 3 mg · kg-1 de méthylprednisolone dissout dans 20 ml de sang autologue. Ľautre patte a reçu la même quantité de sang et fut utilisée comme contrôle. Pendant deux heures du temps ďischémie par tourniquet et 30 minutes après la déflation du tourniquet le sang veineux a été retiré à cinq reprises des deux pattes pour fin de gazométrie (PvO2, PvCO2) pH, acide lactique, créatine pattes pour fin de gazométrie (PvO2, PvCO2) pH, acide lactique, créatine kinase (CK), aspartate aminotransférase (AST) et déhydrogénase lactique (LDH). Durant ľischémie provoquée par le tourniquet, la PvO2 et pH diminuèrent et la PvCO2, ľacide lactique, la CK, ľAST et le LDH augmentèrent progressivement et significativement dans les deux groupes de pattes indiquant une acidose métabolique et respiratoire ainsi qu’une lésion de la cellule musculaire. Cependant, ces changements étaient significativement moindres pour les pattes traitées au methylprednisolone. Ľeffet bénéfique de la méthylprednisolone pourra être attribué à son effet vasodilatateur, son effet stabilisateur de la membrane cellulaire et un effet métabolique direct sur la cellule musculaire squelettique. Même si le temps ďischémie induit par le tourniquet dans notre étude est légèrement différent de la clinique, les résultats suggèrent que les doses élevées de méthylprednisolone peuvent amener un effet bénéfique sur ľischémie provoquée par le tourniquet.

Hospital and Clinical Laboratory Response to Mumps Exposures

Background In response to an outbreak of mumps in the Kansas City area, a multidisciplinary task force quickly formed in our hospital with the goal of protecting patients, families, and employees from the spread of this highly-communicable disease. Methods The clinical laboratory provided proactive mumps IgG testing for all hospital employees. A plan was executed to identify non-immune staff that may be susceptible to mumps. Additionally, the hospital provided immunizations for those employees whose titers were negative. Results Approximately 4,000 blood specimens were drawn from hospital personnel, and immunity was evaluated by measuring mumps-specific antibody. This information assisted in decisions that avoided a significant outbreak in a hospital setting. Conclusion The plan and how it was accomplished, along with costs, successes, and pitfalls, is presented.

The Role of Membrane Phospholipid in Expression of Erythrocyte Rh0(D) Antigen Activity

Abstract Previous investigators have reported that the expression of Rho(D) antigen activity by human erythrocytes and their membranes depended on the presence of phospholipid. In order to further elucidate the role of phospholipids in expression of Rho(D) antigen activity, erythrocyte membranes and partially purified Rho(D) antigens were incubated with bee venom phospholipase A2. Treatment of erythrocyte membranes with phospholipase A2 resulted in loss of Rho(D) antigen activity as detected by hemagglutination inhibition assays. However, subsequent solubilization of these treated membranes with deoxycholate allowed recovery of Rho(D) antigen activity. Phospholipase treatment of solubilized Rho(D) antigens, which had been partially purified by affinity chromatography on anti-Rho(D) IgG agarose columns, did not destroy Rho(D) antigen activity. These results suggested that phospholipids did not affect the antigenic determinants of the Rho(D) antigen since solubilized, partially purified Rho(D) antigens retained their antigenicity following exposure to phospholipase. Phospholipids were presumably required for proper orientation of Rho(D) antigens within erythrocyte membranes since Rho(D) membranes lost their antigenicity following phospholipase treatment.

Enhanced Conditions for DNA Fingerprinting with Biotinylated M13 Bacteriophage

Deoxyribonucleic acid (DNA) fingerprints are Southern blots which have a pattern resembling bar codes. The pattern is created by DNA probes that bind to variable-length repeated sequences of human genomic DNA digested with restriction endonucleases. To improve DNA fingerprints obtained with biotin-labeled M13mp8 replicative form (RF) bacteriophage as the gene probe, the conditions for hybridization and the subsequent washing steps of the filter were refined. Experiments were conducted varying the electrophoresis time, blotting membranes, hybridization solution, and posthybridization washes. The simplicity, sensitivity, and reliability of this nonistopic technique make possible its application for identification of individuals within a species, for parentage testing, and for monitoring bone marrow transplantation.

Identification of Rho(D) antigen in polyacrylamide gels by an enzyme-linked immunoassay.

Human erythrocyte membranes were solubilized in sodium dodecyl sulfate at 100 degree C and subjected to polyacrylamide gel electrophoresis. The gels were sliced into segments and each segment was incubated with anti-Rho(D) IgG, washed, and then incubated with goat anti-human IgG covalently linked to alkaline phosphatase. Para-nitrophenyl phosphate was added to each slice and the absorbance of the solution surrounding each slice was measured at 405 nm. This technique demonstrated that the Rho (D) antigen is a protein with a mol, wt between 13, 000 and 30,000. This method should be applicable to the investigation of other membrane-bound antigens.

Factor V and VIII deficiency treated with therapeutic plasma exchange prior to redo mitral valve replacement.

A 33‐year‐old male was admitted to the hospital for a repeat mitral valve replacement. The original surgery, performed in India in 2008 due to rheumatic heart disease, required massive amounts of plasma replacement during and after the surgery. The patient was admitted to our hospital with extremely low Factor V and Factor VIII activities due to a rare combined Factor V and Factor VIII deficiency. His clinical condition on admission was grave due to severe pulmonary hypertension. It was decided to replace the patients Factor V using therapeutic plasma exchange (TPE) with fresh frozen plasma (FFP) just prior to surgery, and his Factor VIII with Factor VIII concentrate. The patient tolerated the valve replacement surgery very well, without excessive bleeding, and received several more TPE procedures postoperatively. He was successfully made replete with both coagulation factors with little to no bleeding during the procedure and postoperatively. TPE is a promising modality for the treatment of patients with similar factor deficiencies for which a specific factor concentrate is not available, especially those at risk of fluid overload from plasma transfusion. J. Clin. Apheresis 32:196–199, 2017.