Studies on human peripheral blood lymphocytes in vitro. I. Biological and physicochemical properties of the pokeweed mitogen.

The saline extract from the roots of Phytolacca americana (pokeweed) possesses three biological properties; hemagglutinin, leukagglutinin, and mitogen. Fractionation and further purification on calcium phosphate column chromatography revealed that the biologically active substance was eluted in the front moving fraction with 0.05 M phosphate buffer pH 7.5. Analytical separation on polyacrylamide gels in disc electrophoresis yielded a single homogeneous band with an R(f) value of 0.43 containing all three biological activities. This fraction had an ultraviolet absorption spectrum similar to PHA, was stable to both periodate and mercaptoethanol treatment and gave a single band in double diffusion and immunoelectrophoretic analysis against the antibody prepared to the crude PWM saline extract. Absorption studies with red cells or stroma revealed that the hemagglutinin could be selectively removed without significantly altering the mitogen, whereas absorption with leukocytes resulted in loss of both the mitogenic and leukagglutinating activities.

Studies on human peripheral blood lymphocytes in vitro. II. Morphological and biochemical studies on the transformation of lymphocytes by pokeweed mitogen.

A study of the kinetics of RNA and DNA synthesis in PWM-stimulated lymphocytes revealed that RNA synthesis preceded the onset of DNA synthesis by approximately 24 hr and that DNA synthesis and transformation was maximal between 66 to 78 hr. Histochemical and radioautographic studies on PWM stimulated cultures indicated that at 72 hr 50 to 60% of the cell population had been transformed by PWM, and that a distinct cell type bearing cytologic resemblance to the early plasma cell had emerged. The RNA sedimentation profile for newly synthesized RNA in PWM-stimulated cells showed that a large peak of 45 to 50 S material was formed after 24 and 40 hr. PWM thus produces a distinctive transformation of human peripheral blood lymphocytes.

Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

Selective intraoperative cholangiography during laparoscopic cholecystectomy: how selective?

The necessity for intraoperative cholangiography during laparoscopic cholecystectomy has been debated for some time. Numerous retrospective studies favor selective intraoperative cholangiography. Surgeons in favor of the selective policy differ in their personal selective criteria. The aim of this prospective study was to evaluate whether intraoperative cholangiography can be safely omitted during laparoscopic cholecystectomy on all patients who fit a standard set of criteria: normal liver function tests, common bile duct diameter less than 10 mm, and no history of gallstone pancreatitis or jaundice. We undertook a prospective study on 155 consecutive patients treated in a county teaching hospital with symptomatic gallbladder disease who met the above standard set of criteria. One hundred and fifty-five patients meeting these criteria underwent laparoscopic cholecystectomy during a 2-year period from February 1996 through February 1998. Data analyzed included patient history, laboratory and ultrasound findings, operative results, postoperative stay, and intraoperative and postoperative complications. The patients were followed by periodic interviews, physical examination, liver function tests, and/or biliary ultrasound for up to 3 1/2 years with a mean follow-up of 26 months for retained common bile duct stones. Intraoperative cholangiography was performed in only one of the 155 patients studied to confirm common bile duct injury. There were four postoperative complications (2.6%) and one common bile injury (0.6%). Postoperative stay averaged one day. No patients, by history, biliary ultrasound, liver function tests or endoscopy, were found to have retained common bile duct stones during the follow-up period. Our study shows that intraoperative cholangiography is not necessary for patients undergoing laparoscopic cholecystectomy who have normal liver functions tests, common bile duct diameter less than 10 mm, and no history of gallstone pancreatitis or jaundice.

Blood glucose monitoring outside the hospital laboratory.

Glucose was determined with reflectance measurements in an attempt to introduce a quality-control program at a number of primary health care centers. A lyophilized whole blood was used as control material. Experience from two different studies shows that the precision and accuracy were not acceptable. The causes and proposed interventions are discussed.

Simple routine assay for serum urea using immobilized urease.

The method described is a simple routine assay suited for a short series of serum samples. The time needed for one assay is 2 to 3 min from a stand-by arrangement. The urease is immobilized on controlled pore glass. The beads are placed in the column of an enzyme thermistor unit that is part of a continuous flow system. The heat of reaction when urea is degraded to ammonia and carbon dioxide by immobilized urease is measured and recorded continuously. The technique was investigated as regards to flow dependence, linearity, recovery, precision and some possible interfering substances. The within day precision was 0.8% (C.V.) and the day to day precision, during 56 days, was 3.0% (C.V.). Furthermore, the coefficient of correlation between results obtained with the enzyme thermistor unit and a conventional spectrophotometric method was 0.991.

A high-sensitivity immuno-chemiluminescence technique for detection of oligoclonal IgG and IgM in unconcentrated cerebrospinal fluid.

A method is described for detection of oligoclonal IgG and IgM in unconcentrated cerebrospinal fluid (CSF). The proteins were separated by agarose gel electrophoresis and transferred to cellulose nitrate membranes. A double antibody technique with a peroxidase conjugated secondary antibody was used to detect the protein. Bound secondary antibody was visualized by autoradiography using chemiluminescence (ECL) reagent. The procedure presented here allows examination of oligoclonal bands of IgG and IgM in less than 6h. The detection limit for a single band was approximately 10pg and the results were in complete agreement with those achieved by isoelectric focusing (Phast System) and subsequent immunofixation. In 9 of the samples oligoclonal IgM was also detected.

C-reactive protein: the difference between quantitation is serum and EDTA plasma.

We report the differences between using either EDTA plasma or serum in a turbidimetric assay for quantitation of C-reactive protein (CRP). A systematic discrepancy was found for these two sample materials. This was most pronounced in the low concentration range (below 20 mg1(-1)) at which lower values were found in serum than in EDTA plasma. Conversely, in the high concentration range, serum showed slightly higher values. Addition of K3-EDTA to the reaction buffer improved the kinetics for sera with low concentrations of CRP, thus increasing the sensitivity of the assay. We found an overall constant discrepancy of approximately 8% lower values in plasma than in serum (equally for low and high levels of CRP) after the addition of K3-EDTA. The most probable explanation for this effect seems to be the differing water content of serum and EDTA plasma. We discuss the role and function of EDTA in the CRP assay and suggest some hypothetical mechanisms.