Abstracts from the 17th Annual Meeting of the Clinical Cytometry Society

Abstract

Assessment of the proliferative capacity of plasma cells (labelling index) is an important independent prognostic indicator in myeloma. A high labelling index (LI) is associated with bad prognosis or worsening of disease. Conversely, a low index corresponds to a good prognosis. Traditionally, the LI has been determined by a microscope immunofluorescent assay. In recent years, a modified method utilising flow cytometry has been developed. We studied the use of flow cytometry to determine LI in 30 patients with newly diagnosed or relapsed myeloma. Using a combination of CD38 PE and CD138 FITC, and propidium iodide to intercalate double stranded DNA, the DNA content of S phase cells (cycling cells) was calculated using Multicycle software (Phoenix Flow Systems, USA). A cut-off point of 4% was adopted as threshold for significant increase in the LI. Of the 30 patients, 23 (77%) had LI of greater than 4% while 7 (23%) had LI less than 4%. Adoption of flow cytometric method over the traditional manual slide method to determine LI should be encouraged and further studied. It is more objective and much easier to perform as the counting procedure is automated. Studies have found it to correlate well with slide based technique. Whole blood was collected from 7 healthy donors and 8 HIV-1 ϩ individuals (average absolute CD4ϩ T cell counts 875.77, SDϮ 446.61). Standard 4-color flow cytometry techniques were utilized, a total of 20,000 CD8ϩ T cells were acquired for tetramers and the percentage of tetramerϩCD57ϩ cells obtained; for cytokine intracellular assays 50.000 CD4ϩ T cells was collected and the percentage obtained reflected coexpression of IFN-␥ and CD69. Membrane marker acquisition of 40.000 cells was acquired.HIVϩ individuals showed significantly increased CD8ϩCD38ϩ expression and tetramers (Mann-Whitney U test, pϭ0.001; 0.002) in comparison to healthy subjects. HIVϩ individuals demonstrated significantly reduced Absolute CD4ϩ T cell counts pϭ0.004) and CD4ϩ/CD8ϩ ratio (Mann-Whitney U test, pϭ0.001) in comparison to controls. However, assessment of intracellular IFN-␥ in these subjects revealed no significant differences (Mann-Whitney U test, pϾ0.05) compared to controls.This study suggests the existence of homeostatic mechanisms in HIV disease that selectively preserve memory CD8ϩ T cell populations reactive towards ubiquitous pathogens. The preservation of reactive CMV responses may be at the expense of T cell memory to infrequently encountered infectious agents.