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Technical Aspects of the Rosette Tests Used to Detect Human Complement Receptor (B) and Sheep Erythrocyte-Binding (T) Lymphocytes¹

From the Disciplina de Imunologia, Departamento de Microbiologia, Imunologia e Parasitologia da Escola Paulista de Medicina, Caixa Postal 7144, Sào Paulo, Brazil and the Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710

Abstract

The conditions for rosette formation between normal human thymus-derived (T) lymphocytes and unsensitized sheep erythrocytes (E) and between bone marrow-derived (B) lymphocytes and sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC) have been examined in detail in this collaborative study. Mean percentage values for E and EAC obtained at Duke University were 55.4 and 13.3, and at the Escola Paulista de Medicina were 30.4 and 26.2. Eighty-nine to 98% of thymus cells formed E rosettes. Another method for detecting complement receptor lymphocytes utilizing human erythrocytes sensitized with antibody and complement (HEAC) was compared with the EAC indicator. At Duke University the mean percentage value for HEAC was 14.4 and at the Escola Paulista de Medicina that value was 18.3. The variation in rosette percentages when the same normal donor was tested on different days and the effect of storage of blood and/or lymphocytes on rosette formation were also examined. Rosette formation between T lymphocytes and E was found to be temperature dependent, with maximum values occurring between 10°C and 25°C with no rosette formation occurring at O°C or 37°C. Some thymocytes, however, were capable of forming E rosettes at 37°C. When EAC peripheral blood lymphocyte rosettes were determined below 37°C, some T lymphocytes were able to form rosettes with E receptors on the EAC indicator cells. Use of HEAC instead of EAC eliminated the interference of E rosette formation with the latter indicator. When centrifugation was compared with rotation in the procedures to detect complement receptor lymphocytes, it was found that fewer B cells were detected when the centrifugation procedure was omitted. This paper further demonstrates the use of rosette formation and gradient centrifugation for the specific depletion of B or T cells from peripheral blood lymphocytes.

Footnotes

¹ This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil and by Research Grant AM 08054 from the National Institutes of Health.

² From the Escola Paulista de Medicina, Sao Paulo, Brazil.

³ From Duke University Medical Center, Durham, North Carolina 27710.

⁴ Address reprint requests to: Dr. Richard S. Metzgar, Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.

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