Use of fluorescence threshold triggering and high-speed flow cytometry for rare event detection.

A simple rare event detection method utilizing dual-parameter flow cytometry is described, which allows quantitation of specific cellular events at the level of two cells in 10(7) total cells. Using a standard unmodified single laser flow cytometer sampling at a rate of 25,000 events/sec and a fluorescence discriminator, 10(7) total cells are processed in 7 min. The assay involves precise characterization of instrument flow rates to calculate total events processed by the cytometer rather than accumulate total events in computer memory. This method of detecting rare events is demonstrated by using a model system of breast cancer cells labeled with a metabolically activated dye and serially diluted into normal peripheral blood. Potential applications include validation of methods to detect minimum residual disease following myeloablative therapy, detection of any remaining tumor cells following purging methods, and validation of methods to detect circulating fetal cells in maternal blood.

Quantitation of T cell depletion by limiting dilution analysis.

BACKGROUND: We evaluated a culture method for enumeration of residual T cells remaining in marrow after treatment with antibody and complement or with immunotoxin. METHODS: Marrow cells were cultured at limiting dilutions with phytohemagglutinin in the presence of Epstein Barr virus transformed human lymphoblastoid cells, and supernates were tested three days later for IL-2 by a cell proliferation assay. This method provides a simple, reliable, objective and rapid enumeration of T cells in marrow before and after treatment. RESULTS: Approximately 6% of untreated marrow mononuclear cells can produce IL-2 in such clonal cultures. Treatment with antibodies and complement under conditions identical to those used for our previous clinical trials produced a 3.7 log depletion of IL-2 precursors, whereas treatment with a ricin A chain anti-CD3 immunotoxin produced a 3.0 log depletion. CONCLUSIONS: Clinical correlations are in progress for assessing whether T cell depletion evaluated with the present method equals previous techniques. The extreme depletion of T cells accomplished by these methods may partly account for the high graft failure rate seen in our clinical trials.

In vitro comparison of two methods of T cell depletion associated with different rates of graft failure after allogeneic marrow transplantation.

Clinical trials in another center have shown a substantially lower risk of graft failure associated with T cell depletion by treatment of donor marrow with the use of an antibody against CD6 compared to depletion with a mixture of eight antibodies previously used for clinical trials in our center. In order to evaluate mechanisms possibly responsible for this difference, we compared lymphoid cell surface phenotypes and in vitro functions in marrow cells treated by complement-mediated lysis with anti-T12 (CD6) or with the eight antibody mixture. Treatment with the eight antibody mixture produced more than three log depletion of precursors for IL-2-producing cells (pIL-2) and approximately one log depletion of precursors for NK cells. On the other hand, treatment with anti-T12 produced approximately one log depletion of pIL-2 and had no effect on NK precursors. Additional studies were carried out with treated marrow cells cultured in medium containing recombinant IL-2. Compared to cells treated with the eight antibody mixture, the marrow cells that remained after anti-T12 treatment had more cytotoxic activity against K562, Daudi and an EBV-transformed human B cell line during the first 6 days of culture, but marrows treated by the two methods showed similar cytotoxic activity after 10 days of culture. Cultures from marrow treated with anti-T12 contained more CD3+ and CD6+ cells than cultures from marrow treated with the eight antibody mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo inhibition of neutrophil function in the rabbit using monoclonal antibody to CD18.

The CD11b/CD18 membrane antigen complex has been shown to be essential for normal neutrophil function in vitro, and patients lacking this antigen exhibit severe neutrophil dysfunction. Murine monoclonal antibody 60.3 (Ab 60.3), directed toward CD18, is a potent inhibitor of human neutrophil function in vitro. To determine whether Ab 60.3 might similarly inhibit neutrophil function in vivo, we measured the effect of antibody administration on neutrophil localization to polyvinyl sponges placed s.c., in rabbits. Studies in 25 animals showed that infusion of Ab 60.3 at the time of sponge insertion resulted in dose-dependent inhibition of in vivo neutrophil migration with almost complete paralysis of neutrophil migration at higher antibody doses. These studies confirm the functional relevance of CD18, demonstrate that neutrophil function can be profoundly inhibited in vivo by Ab 60.3, and suggest that substances such as AB 60.3 may be clinically useful as potent anti-inflammatory agents.

Phase 1 immunolymphoscintigraphy with an In-111-labeled antimelanoma monoclonal antibody.

A phase 1 study was conducted using a monoclonal antimelanoma antibody-DTPA conjugate labeled with indium-111, for immunolymphoscintigraphy in patients with metastatic melanoma. The imaging agent, labeled with 1 mCi (37 MBq) In-111, was administered as an interstitial interdigital injection to six patients scheduled to undergo lymph node dissection for suspected metastatic malignant melanoma. No adverse effects were observed in any of the patients either during or after the infusion as determined by clinical and laboratory parameters. Antibodies to the murine immunoglobulin were produced in some patients. Regional lymph nodes were visualized whether tumor-bearing or not, and light microscopic autoradiography showed In-111 activity associated with histiocytes. One of two patients harboring both tumor-bearing and tumor-free lymph nodes exhibited preferential localization in tumor-bearing nodes. The authors conclude that this study demonstrates safety of the radiopharmaceutical and that further study is needed to improve its usefulness for diagnosis of lymph node metastases.

A new method with high sensitivity and specificity for localization of abnormal parathyroid glands.

A novel method for localization of abnormal parathyroid glands involving color-processing of nuclear scintigrams of the neck after injection of Thallium-201 and Technetium pertechnetate is presented with surgical correlation. Preoperative localization of single parathyroid adenomas was successful in 88% of previously unoperated patients and in 85.7% of those with adenomas not located at previous surgery. Eighty-three per cent of glands with secondary hyperplasia, 66% of glands with primary hyperplasia, and one carcinoma were localized. No abnormal studies were seen in non-hyperparathyroid hypercalcemia, and no false positive studies were seen. Localization appeared related to larger adenomas (300-5000 mg), although one of 60 mg was localized. Color-comparison dual-isotype scintigraphy was useful for localization of parathyroid adenomas and hyperplastic glands and exceeded the reported sensitivity of either ultrasonography or computerized tomography. It deserves wider evaluation in preoperative management of at least hyperparathyroidism of the primary or persistent types.