A. Michael Zimmer

Reinfusion of postoperative wound drainage in total joint arthroplasty. Red blood cell survival and coagulopathy risk

Fifty patients with total joint arthroplasties (28 total hip arthroplasties, 11 total knee arthroplasties, and 11 bilateral total knee arthroplasties) received autotransfusions from their postoperative wound drainage. The blood was collected in a closed sterile drainage system without any additional anticoagulant. Pre- and postoperative measurements were made of the patients hemoglobin, platelets, fibrinogen, haptoglobin, fibrin degradation products, and D-dimer (a specific type of fibrin degradation product). Red blood cell survival was assessed in 16 of the patients by labeling the shed blood with 51Cr sodium chromate prior to reinfusion. To control for fluid shifts, continued bleeding, and dilution effects of further transfusions in the immediate postoperative period, 10 patients also had their native blood labeled with 111In oxime. In this study, the mean estimated blood loss was 1,062 mL (+/- 1,247) with a mean wound drainage of 836 mL (+/- 338). Of this, a mean of 450 mL (+/- 261) of blood was was given back to the patient in addition to routine, preoperative autologous donated blood. Six (12%) patients experienced transient fevers at the time of retransfusion. Detailed hematologic studies were performed on the shed blood in 19 patients. The collected blood was completely defibrinated, but did contain fibrin degradation products, as indicated by the D-dimer level, and hemolyzed blood as the haptoglobin was reduced. Even though the blood containing the above breakdown products was reinfused to the patients, there were no clinical manifestations of disseminated intravascular coagulation. Both the hemolyzed and defibrinated products were subsequently cleared by the body.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid miniaturized chromatography for Tc-99m IDA agents: comparison with gel chromatography.

Miniaturized chromatographic systems for determining free pertechnetate and hydrolyzed reduced 99mTc levels in comericial 99mTc-labeled iminodiacetate (IDA) hepatobiliary radio-pharmaceuticals were evaluated and the results compared with gel chromatography column scanning (GCS). Commercial IDA agents were evaluated including 2,6-dimethyl IDA, p-isopropyl IDA, p-butyl IDA, and 2,6-diisopropyl IDA. Of all the chromatography systems evaluated, only Gelman ITLC-SA with 20% NaCl and Gelman ITLC-SG with distilled water correlated with GCS in evaluating free pertechnetate and hydrolyzed reduced 99mTc levels for all IDA radiopharmaceuticals. The miniaturized chromatography procedure, as outlined, is rapid, taking less than 4 min, and can easily be incorporated into the daily quality control program in any nuclear medicine facility.

Human IgE, IgG and IgA Antibody Responses to T101, a Murine Monoclonal Antibody against Human Lymphocytes: Implications for Pathogenesis, Risk and Avoidance of Adverse Immunologic Reactions

To study immune responses that may play a role in immediate-type allergic reactions (ITAR) and serum-sickness-like reactions that have been reported with administration of monoclonal antibodies (MAb), we measured by enzyme-linked immunosorbent assay serum levels of IgE, IgG, and IgA human antimurine antibody (HAMA) to T101, a murine IgG2a antibody that has been used in the treatment of cutaneous T cell lymphoma (CTCL) and chronic lymphocytic leukemia (CLL). None of 8 patients (4 with CLL, 4 CTCL) pretreated with T101 had elevated titers of any HAMA class tested as compared to normal control sera. All CTCL patients who had elevated total serum IgE levels, but normal total serum levels of other antibody classes, had significant rises in IgE, IgG, and IgA HAMA to T101 after intravenous MAb infusion. However, the CLL patients who were hypogammaglobulinemic failed to develop significant rises in HAMA. Three patients (2 CLL, 1 CTCL) had ITAR (e.g., urticaria, angioedema, bronchospasm) associated with infusion of T101; prophylactic medication regimens based upon the control of radiographic contrast media reactions were used with apparent benefit in subsequent infusions in these patients. All 8 patients had negative immediate intradermal skin tests (1 microgram/ml) to T101 prior to its infusion. Our data confirm that (1) non-IgE-mediated mechanisms cause ITAR from this MAb, possibly by a mechanism inherent in the action of this MAb against lymphocytes, and (2) that isotypic antibody responses to MAb vary with the type of malignancy being treated.(ABSTRACT TRUNCATED AT 250 WORDS)

Internalization and shedding of Lym-1 monoclonal antibody following interaction with surface antigens of a cultured human B cell lymphoma

Modulation of surface antigens of Raji Burkitts lymphoma cells by monoclonal antibody Lym-1 was investigated by flow cytometry and radioimmunoassay (RIA). Raji cells, treated with antibody conjugated with FITC, became bright green as determined by FACS and the FITC-labeled Lym-1 remained associated with target cells for up to 48 hr after incubation at either 4 or 37 degrees C. Lym-1 antibody linked with biotin also bound to Raji cells and rendered these cells highly reactive with avidin-phycoerythrin (APE). However, the APE fluorescence intensity measured by FACS decreased substantially when Raji cells were cultured at 37 degrees C for 1 hr prior to APE exposure but not when incubation was carried out at 4 degrees C, indicating a disappearance of antibody from the surface of the metabolically active cells. This process was time dependent with a total loss of surface-bound biotinylated antibody occurring over a period of approximately 2 hr. Raji cells exposed to both fluoresceinated and biotinylated Lym-1 in a double labeling experiment became positive to both reagents. The flow cytometric profile was not altered when these cells were incubated for 1 hr at 4 degrees C followed by reaction with APE. However, they failed to react with APE when the 1-hr incubation took place at 37 degrees C despite the fact that they remained FITC positive, suggesting that the antibody with its fluorescent label had entered the cells. Utilizing 131I-labeled Lym-1 it was determined that approximately 50% of initially bound antibody had dissociated from the cells within the first 2 hr of incubation at 37 degrees C, although the remainder persisted with targets for up to 48 hr. The HPLC protein profile indicated that the radioactivity found in the culture supernatants and cytoplasm was associated with whole antibody, degradation products, and Ig complexes with antigen. Therefore, the present findings suggest that Lym-1 Ig molecules react with cell surface antigens and are rapidly internalized and shed, resulting in the disappearance of antibody from the surface membrane of Raji cells.

Rapid miniaturized chromatography for 111In labeled monoclonal antibodies: Comparison to size exclusion high performance liquid chromatography

Our laboratory investigated the use of a rapid miniaturized chromatography system, ITLC-SG with 0.9% NaCl, to assess the radiochemical purity of 111In labeled monoclonal antibodies (MoAbs). Radiochemical analysis was performed on numerous 111In labeled antibody preparations with labeling efficiencies ranging from 40 to greater than 95% and the results compared to those obtained with size exclusion high performance liquid chromatography (HPLC). The chromatographic procedure involved challenging radiolabeled antibodies with 0.05 M DTPA to chelate unbound and/or non-specific bound 111In, spotting on miniaturized instant thin layer-silica gel chromatography strips, developing in 0.9% NaCl, and counting appropriate segments for radioactivity. Results of the study demonstrated that the miniaturized chromatography procedure was rapid, taking less than 4 min to complete, and accurate in assessing the amount of unbound or non-specific bound 111In in 111In labeled monoclonal antibodies, when compared to size exclusion HPLC.

Stability of radioiodinated monoclonal antibodies: In vitro storage and plasma analysis

The in vitro stability and immunointegrity of four radioiodinated monoclonal antibodies was evaluated in various storage conditions and also in plasma samples. The monoclonal antibodies studied included T101, B72.3, Lym1, and 16.88. Stabilities of typical monoclonal antibody therapy solutions, with radioactivities ranging from 2220 to 3700 MBq (60-100 mCi) were assessed using conventional instant thin layer chromatography and size exclusion high performance liquid chromatography. Radioimmunoreactivity was assessed using a live cell attenuated cell, or mucin-linked bead assay. Results of the study demonstrated that therapy solutions were stable to degradation, if properly stored in 5 or 10% human serum albumin at 4 degrees C for the duration of the study (5 days). Minor losses in immunoreactivity were also measured in stabilized therapy solutions. When incubated in plasma samples, radioiodinated monoclonal antibodies generally remained stable for the duration of the study (3 days). However, significant decreases in immunoreactivity were measured for specific radioiodinated monoclonal antibody preparations.

Considerations for tomographic imaging of monoclonal antibodies

Monoclonal antibodies have begun to assume a significant role in clinical research. The ability to label these agents has initiated research in the areas of radioimmunodetection and radioimmunotherapy. In the case of antibodies directed against tumor antigens, imaging has been employed to help assess location and extent of disease, and to provide information and extent of disease, and to provide information concerning biodistribution to be used in subsequent dosimetric calculations. Because of the low counting statistics characteristic of such images, the use of single photon emission computed tomography (SPECT) is suggested as a potential method of improving the diagnostic yield from image data. Careful attention to acquisition parameters and image processing options is needed if these goals are to be achieved.

Nuclear medicine imaging workstations based on personal computer technology

The development of personal computer technology has resulted in extremely powerful, inexpensive computers available as consumer items. With the addition of suitable hardware for gamma camera interfacing and image display, such systems can be transformed into fully functional nuclear medicine computers capable of performing all of the acquisition and processing tasks required in a modern radioisotope imaging department. Such an approach to nuclear medicine computerization offers many advantages in terms of flexibility, speed, cost, and expandability.

Leucocyte labeling with colloidal 111In in whole blood

A simple method of 111In leucocyte labeling in whole blood is described. 111In-colloid is initially formulated using iron (50 micrograms Fe+3). 111In-colloid is then added to 10mL of whole blood and leucocyte labeling is achieved by phagocytosis. Non-phagocytosed 111In-colloid is solubilized using a sodium citrate solution. Labeling efficiencies of 60-80% have been achieved with this method. If needed, excess 111In-soluble-complexes can be removed in the plasma fraction by appropriate centrifugation. The labeling method described is easy to use and has the advantage of selectively labeling leucocytes in a whole blood mixture.